Recombinant super-compound interferon and uses thereof

ABSTRACT

This invention provides a method for treating tumors in a subject, comprising administering to the subject an effective amount of a recombinant interferon, wherein the interferon has the amino acid sequence of SEQ ID NO:2 and is encoded by the nucleotide sequence SEQ ID NO:1, wherein the tumors are selected from the group consisting of skin cancer, basal cell carcinoma, liver cancer, thyroid cancer, rhinopharyngeal cancer, solid carcinoma, prostate cancer, esophageal cancer, pancreatic cancer, superficial bladder cancer, hemangioma, epidermoid carcinoma, cervical cancer, glioma, leucocythemia, acute leucocythemia, chronic leucocythemia, lymphadenoma, and polycythemia vera.

The application is a continuation application of U.S. Ser. No.12/246,153, filed Oct. 6, 2008 which is a continuation application ofU.S. Ser. No. 11/077,813, filed Mar. 10, 2005, which is acontinuation-in-part of U.S. Ser. No. 10/927,975, filed Aug. 26, 2004,which is a continuation-in-part of U.S. Ser. No. 10/650,365, filed Aug.28, 2003, which is a continuation-in-part of Int'l App'l No.PCT/CN02/00128, filed Feb. 28, 2002, which claims priority of ChineseApp'l No. 01104367.9, filed Feb. 28, 2001. The contents of the aboveapplications are hereby incorporated in their entireties by referenceinto this application.

Throughout this application, various publications are referenced.Disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention is related to a field of bioengineering. Specificallythis invention relates to a recombinant super-compound interferon(rSIFN-co) or its equivalent with changed spatial configuration, highefficacy and low side effects. Therefore, high dose of rSIFN-co may beused. This invention also relates to a process to produce saidsuper-compound interferon (rSIFN-co) or a pharmaceutical compositioncomprising said super-compound interferon (rSIFN-co) or its equivalent,and uses of said interferon or composition for anti-viral and anti-tumortherapy.

BACKGROUND OF THE INVENTION

IFN-con is a new interferon molecule constructed with the most popularconservative amino acid found in natural human IFN-α subtypes usinggenetic engineering methods. U.S. Pat. Nos. 4,695,623 and 4,897,471 havedescribed it. IFN-con had been proven to have broad-spectrum IFNactivity and virus- and tumor-inhibition and natural killer cellactivity. U.S. Pat. No. 5,372,808 by Amgen, Inc. addresses treatmentInfergen® (interferon alfacon-1). Chinese Patent No. 97193506.8 byAmgen, Inc. addresses re-treatment of Infergen® (interferon alfacon-1)on hepatitis C. Chinese Patent No. 98114663.5 by Shenzhen JiushengBio-engineering Ltd. addresses recombinant human consensus interferon-αtreatment for hepatitis B and hepatitis C.

The United States Food and Drug Administration (FDA) authorized Amgen toproduce Infergen® (interferon alfacon-1) with E. Coli. for clinicalhepatitis C treatment at the end of 1997.

Hepatitis B patients can be identified when detecting HBsAg and theHBeAg. IFN-α is commonly used in clinics to treat hepatitis B. IFN-αbinds superficial cell membrane receptors, thus inhibiting DNA and RNA(ribonucleic acid) duplication and inducing some enzymes to preventduplication of the virus in hepatitis-infected cells. All IFNs caninhibit DNA duplication of viruses, but they cannot inhibit the e and santigen expression.

An outbreak of atypical pneumonia, referred to as severe acuterespiratory syndrome (SARS) and first identified in Guangdong Province,China, has spread to several countries. Similar cases were detected inpatients in Hong Kong, Vietnam, and Canada from February and March 2003.The World Health Organization (WHO) issued a global alert for theillness. In mid-March 2003, SARS was documented in health care workersand household members who had cared for patients with severe respiratoryillness in the Far East. Many of these cases could be traced throughmultiple chains of transmission to one health care worker from GuangdongProvince who visited Hong Kong, where he was hospitalized with pneumoniaand died. By late April 2003, thousands of SARS cases and hundreds ofSARS-related deaths from over 25 countries around the world werereported to WHO. Most of these cases occurred through exposure to SARSpatients in household or health care settings. This invention provides amethod to prevent and/or treat SARS. This disclosure describesrecombinant super-compound interferon (rSIFN-co), method to produce thesame and uses thereof. Particularly, the super-compound interferondisclosed herein is capable of inhibiting, preventing and/or treatingthe hepatitis viruses, SARS virus, or virus-induced upper respiratorydiseases, the Influenza virus, for example Avian Influenza virus andEbola virus.

In addition, rSIFN-co is effective in preventing and/or treating viraldiseases and tumors with less side effects as compared to otheravailable interferons.

SUMMARY OF THE INVENTION

This invention provides a recombinant super-compound interferon(rSIFN-co) and its equivalent with changed spatial configuration, highefficacy and low side effects. Therefore, high dose of rSIFN-co may beused.

This invention also provides artificial gene encoding for thesuper-compound interferon or its equivalent.

This invention provides a vector comprising the gene which codes for thesuper-compound interferon or its equivalent.

This invention provides an expression system comprising the vectorcomprising the gene which codes for the super-compound interferon or itsequivalent. This invention also provides a host cell comprising thevector comprising the gene which codes for the recombinantsuper-compound interferon (rSIFN-co) or its equivalent. Said host cellmay be eukaryotic or prokaryotic, such as E. Coli.

This invention provides a method for producing a recombinantsuper-compound interferon (rSIFN-co) with changed spatial configurationand enhanced antiviral activity comprising steps of:

-   (a) Introducing nucleic acid molecule which codes for said    interferon with preferred codons for expression to an appropriate    host; and-   (b) Placing the introduced host in conditions allowing expression of    said interferon.

This invention provides the method for producing recombinantsuper-compound interferon (rSIFN-co), further comprising recovery of theexpressed interferon.

This invention provides a method for inhibiting, preventing or treatingviral diseases, or for inhibiting or treating tumors in a subjectcomprising administering to the subject an effective amount of thesuper-compound interferon or its equivalent.

This invention provides the above-described method whereinsuper-compound interferon is administered orally, via vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal ormucosal administration, or by inhalation via a respirator.

This invention provides the method to prevent or treat viral diseaseswherein the viral diseases is hepatitis A, hepatitis B, hepatitis C,other types of hepatitis, infections of viruses caused by Epstein-Barrvirus, Human Immunodeficiency Virus (HIV), Ebola virus, Severe AcuteRespiratory Syndrome Virus (SARS), Influenza virus, Cytomegalovirus,herpes simplex viruses, or other types of herpes viruses, papovaviruses,poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cellleukemia viruses I, or human T-cell leukemia viruses II, or human T-cellleukemia virus III.

This invention provides the method to prevent or treat viral diseaseswherein the viral diseases are Human Immunodeficiency Virus (HIV) andEbola virus.

This invention provides a method for anti-hepatitis activities. It caninhibit HBV-DNA replication, HBsAg and HBeAg production.

This invention provides a method to prevent or treat upper respiratoryinfection diseases.

This invention provides a method to prevent or treat tumors or cancerswherein the tumor is skin cancer, basal cell carcinoma and malignantmelanoma, renal cell carcinoma, liver cancer, thyroid cancer,rhinopharyngeal cancer, solid carcinoma, prostate cancer,stomach/abdominal cancer, esophageal cancer, rectal cancer, pancreaticcancer, breast cancer, ovarian cancer, and superficial bladder cancer,hemangioma, epidermoid carcinoma, cervical cancer, non-small-cell lungcancer, small-cell lung cancer, glioma, leucocythemia, acuteleucocythemia and chronic leucocythemia, chronica myelocytic leukemia,hairy cell leukemia, lymphadenoma, multiple myeloma, polycythemia vera,or Kaposi's sarcoma.

This invention provides a method for preventing or treatingvirus-induced diseases in a subject comprising administering to thesubject an effective amount of recombinant super-compound interferon ora functional equivalent thereof.

The super-compound interferon (rSIFN-co) may be administered orally, viavein injection, muscle injection, peritoneal injection, subcutaneousinjection, nasal or mucosal administration, or by inhalation via arespirator.

This invention provides a method for inhibiting the causative agent ofvirus-induced diseases, comprising contacting the causative agent withan effective amount of super-compound interferon or its equivalent.

This invention also provides a method for inhibiting virus-induceddiseases, comprising contacting an effective amount of thesuper-compound interferon with said virus or cells. This contact couldbe direct or indirect.

This invention provides a composition comprising an effective amount ofthe super-compound interferon capable of inhibiting, preventing ortreating virus-induced diseases, and a suitable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the recombinant super-compound interferon capable ofinhibiting, preventing or treating virus-induced diseases in a subject,and a pharmaceutically acceptable carrier.

This invention provides a method for preventing or treating tumors in asubject comprising administering to the subject an effective amount ofrecombinant super-compound interferon or a functional equivalentthereof.

This invention provides a method for inhibiting tumors, comprisingcontacting the causative agent with an effective amount ofsuper-compound interferon or its equivalent.

This invention also provides a method for inhibiting tumors, comprisingcontacting an effective amount of the super-compound interferon withsaid virus or cells. This contact could be direct or indirect.

This invention provides a composition comprising an effective amount ofthe super-compound interferon capable of inhibiting, preventing ortreating tumors, and a suitable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the recombinant super-compound interferon capable ofinhibiting, preventing or treating tumors in a subject, and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. rSIFN-co cDNA sequence designed according to E. Coli. codonusage and deduced rSIFN-co amino acid sequence

FIGS. 2A-B. Sequence of another super-compound interferon

FIG. 3. Diagram of pLac T7 cloning vector plasmid

FIG. 4. Diagram of pHY-4 expression vector plasmid

FIG. 5. Construction process of expression plasmid pHY-5

FIG. 6-A. Circular Dichroism spectrum of Infergen®

(Tested by Analysis and Measurement Center of Sichuan University)

Spectrum range: 250 nm-190 nmSensitivity: 2 m°/cmLight path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml IFN-con1, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

Infergen® (interferon alfacon-1), made by Amgen Inc., also known asconsensus interferon, is marketed for the treatment of adults withchronic hepatitis C virus (HCV) infections. It is currently the onlyFDA-approved, bio-optimized interferon developed through rational drugdesign and the only interferon with data on the label specifically fornon-responding or refractory patients. InterMune's sales forcere-launched Infergen® in January 2002 with an active campaign to educateU.S. hepatologists about the safe and appropriate use of Infergen®,which represents new hope for the more than 50 percent of HCV patientswho fail other currently available therapies.

FIG. 6-B. Circular Dichroism spectrum of Infergen® From Reference[Journal of Interferon and Cytokine Research. 16:489-499 (1996)]

Circular dichroism spectra of concensus interferon subforms. Concensusinterferon was fractionated using an anion exchange column. Samples weredialyzed into 10 mM sodium phosphate, pH 7.4. Measurements were made onJasco J-170 spectopolarimeter, in a cell thermostat at 15° C. (—),acylated form; (--) cis terminal form; ( . . . ), met terminal form. A.Far UV Spectrum. B. Near UV Spectrum.

FIG. 6-C. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 320 nm-250 nmSensitivity: 2 m°/cmLight path: 2 cm

Equipment: Circular Dichroism J-500C

Samples: contains 0.5 mg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

FIG. 6-D. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 250 nm-190 nmSensitivity: 2 m°/cmLight path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

Clearly, as evidenced by the above spectra, the secondary or eventertiary structure of rSIFN-co is different from Infergen®.

FIG. 7. rSIFN-co Crystal I

FIG. 8. rSIFN-co Crystal II

FIG. 9. The X-ray Diffraction of rSIFN-co Crystal

FIG. 10. Comparison of Inhibition Effects of Different Interferons onHBV Gene Expression

FIG. 11A-C. Recombinant Super-Compound Interferon Spray

Height: 90 mm

Width: 25 mm (bottom), 6 mm (top)

Weight: 9 g

Volume delivery: 0.1 ml

FIG. 11D. Recombinant Super-Compound Interferon Spray

When using the spray for the first time, take off the cap and dischargein the air several times until some liquid squirts out. Do not need totest spray for subsequent uses. To use, follow the illustrations shownin the figure, i.e.: (1) Pre-spray and (2) Press down on the nozzle torelease the medication.

FIG. 12. Comparison of Anti-SARS Activity of Interferons: Left top panelis negative control i.e. no virus added. Right top panel is positivecontrol i.e. virus is added, but no rSIFN-co added. Left bottom panel isrSIFN-co with SARS Virus. Right bottom panel is αIFN.

FIGS. 13A-1. Curves of Changes of Body Temperature in Group A (5patients)

This figure is the record of body temperature changes of 5 patients inGroup A.

FIGS. 13A-2. Curves of Changes of Body Temperature in Group A (5patients)

This figure is the record of body temperature changes of the other 5patients in Group A.

FIGS. 13B-1. Curves of Changes of Body Temperature in Group B (5patients)

This figure is the record of body temperature changes of 5 patients inGroup B.

FIGS. 13B-2. Curves of Changes of Body Temperature in Group B (6patients)

This figure is the record of body temperature changes of the other 6patients in Group B.

FIG. 14. Graph of Inhibition of Wild-Type HIV by rSIFN-co using EXCELand Luciferase as Y axis and concentration of rSIFN-co as X axis. Aclear inverse dose-dependent response has been shown.

FIG. 15. Graph of Inhibition of Drug Resistant HIV by rSIFN-co usingEXCEL and Luciferase as Y axis and concentration of rSIFN-co as X axis.A clear inverse dose-dependent response has been shown.

FIG. 16. rSIFN-co Inhibition of Influenza Virus: on the left, thecontrol well is shown with Influenza virus added and without interferon,the cells had obvious CPE, such as rounding of cells, cell necroses,decrease in reflective light and sloughing off.

On the right, the experimental wells is shown containing Influenza virusand rSIFN-co at concentration 10 nanogram per milliliter (ng/ml) hadmorphology comparable to normal cells.

FIGS. 17A-H. Clinical Report of a patient with mammary and ovariancancers. These figures show an obvious anti-cancer effect of rSIFN-co onthis patient.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant Super-Compound Interferon (rSIFN-co)

This invention provides a recombinant super-compound interferon(rSIFN-co) or an equivalent thereof with changed spatial configuration.This invention reveals that proteins with the same primary sequencemight have different biological activities. As illustrated in thisapplication, proteins with identical amino acid sequences may havedifferent activities. The efficacy of these proteins may sometimes beimproved and, sometimes, proteins with changed spatial configurationwould reveal new function.

As defined herein, equivalents are molecules which are similar infunction to the compound interferon. An equivalent could be a deletion,substitution, or replacement mutant of the original sequence.Alternatively, it is also the intention of this invention to covermimics of the recombinant super-compound interferon (rSIFN-co). Mimicscould be a peptide, polypeptide or a small chemical entity.

The recombinant super-compound interferon (rSIFN-co) described hereinincludes but is not limited to interferon α, β, γ or ω. In anembodiment, it is IFN-1α, IFN-2β or other mutants.

In another embodiment, the recombinant super-compound interferon(rSIFN-co) disclosed has higher efficacy than α, β, γ, ω or acombination thereof and as compared to the interferons disclosed in U.S.Pat. Nos. 4,695,623 and 4,897,471. This recombinant super-compoundinterferon (rSIFN-co) is believed to have unique secondary or tertiarystructure, wherein the 3-dimensional change is the result of changes inits production process. (See e.g. FIG. 6.)

The recombinant super-compound interferon (rSIFN-co) described hereinhas spatial structure change(s) resulting from the changes of itsproduction process.

Lower Side Effects

The recombinant super-compound interferon (rSIFN-co) possesses lowerside effects when compared with other interferons. These lower sideeffects allow for higher dosages to be used on patients in need ofinterferon treatments. These lower side effects open the possibility ofusing rSIFN-co for prevention and/or treatment of other diseases.Accordingly, this invention provides the recombinant super-compoundinterferon (rSIFN-co) with less side effects when administered to asubject.

This invention provides recombinant super-compound interferon (rSIFN-co)with less side effects as compared to all currently availableinterferons.

This invention further provides a method for treating or preventingviral diseases or tumors in a subject comprising administering to thesubject an effective amount of the rSIFN-co with less side effects ascompared to all currently available interferons. Therefore, high dose ofrSIFN-co may be used. In an embodiment, the effective amount ofrecombinant super-compound interferon is in nanogram level.

Process to Produce rSIFN-co

Artificial Gene

This invention also provides artificial gene encoding for thesuper-compound interferon or its equivalent. It is within the ordinaryskill to design an artificial gene. Many methods for generatingnucleotide sequence and other molecular biology techniques have beendescribed previously. See for example, Joseph Sambrook and David W.Russell, Molecular Cloning: A laboratory Manual, December 2000,published by Cold Spring Harbor Laboratory Press.

The recombinant super-compound interferon (rSIFN-co) may also beproduced with its gene as artificially synthesized cDNA with adjustmentof its sequence from the wild-type according to codon preference of E.Coli. Extensive discussion of said codon usage (preference) may be foundin U.S. Pat. No. 4,695,623. See e.g. column 6, line 41—column 7, line35.

Vector

This invention provides a vector comprising the gene which codes for thesuper-compound interferon or its equivalent.

This invention provides an expression system comprising the vectorcomprising the gene which codes for the super-compound interferon or itsequivalent. The cells include, but are not limited to, prokaryotic oreukaryotic cells.

This invention also provides a host cell comprising the vectorcomprising the gene which codes for the recombinant super-compoundinterferon (rSIFN-co) or its equivalent.

This invention provides a method for producing a recombinantsuper-compound interferon (rSIFN-co) with changed spatial configurationand enhanced antiviral activity comprising steps of:

-   (a) Introducing nucleic acid molecule which codes for said    interferon with preferred codons for expression to an appropriate    host; and-   (c) Placing the introduced host in conditions allowing expression of    said interferon.

This invention provides the method for producing recombinantsuper-compound interferon (rSIFN-co), further comprising recovery of theexpressed interferon.

Expression System

The above-described recombinant super-compound interferon (rSIFN-co) maybe produced by a high-efficiency expression system which uses a specialpromoter, enhancer or other regulatory element. In an embodiment thepromoter is inducible. Said inducible promoter includes but is notlimited to P_(BAD), heat shock promoters or heavy metal induciblepromoters. Heat shock promoters are activated by physical means, whileother promoters are activated by chemical means, for example IPTG orTetracyclin. IPTG is added to the cells to activate the downstream geneor removed to inactivate the gene. Tetracyclin is used to inducepromoters or to regulate the strength of promoters.

In an embodiment the promoter is P_(BAD). Since early nineties, theproperties of the mechanism of expression and repression of P_(BAD) byAraC have been studied extensively, and their interactions have beendissected at the molecular level. See Schleif, R. S. 1992 DNA looping.Annu. Rev. Biochem. 61:199-223. The AraC protein is both a positive andnegative regulator, when present, it turns on the transcription from theP_(BAD) promoter, when absent, the transcription occurs at a very lowrate. See Guzman, L. M. et al. (1995) J. Bact. 177: 4121-4130. Theefficacy and mechanism of P_(BAD) promoter is well known by otherordinary skilled artisans and is commercially-available.

The commercially-available Invitrogen expression kit includes pBADvectors' designed to provide precise control of expression levels. ThearaBAD promoter initiates gene expression. It's both positively andnegatively regulated by the product of the araC gene, a transcriptionalregulator that forms a complex with L-arabinose. In the absence ofarabinose, the AraC dimer contacts the O2 and I1 half sites of thearaBAD operon, forming a 210 bp DNA loop. For maximum transcriptionalactivation, two events are required: first, Arabinose binds to AraC. Theprotein releases the O2 site and binds the I2 site, which is adjacent tothe I1 site. This releases the DNA loop and allows transcription tobegin. Second, the cAMP activator protein (CAP)-cAMP complex binds tothe DNA and stimulates binding of AraC to I1 and I2. Basal expressionlevels can be repressed by introducing glucose to the growth medium.Glucose acts by lowering cAMP levels, which in turn decreases thebinding of CAP. As cAMP levels are lowered, transcriptional activationis decreased. Invitrogen's pBAD vectors are specifically designed formaximum expression and ease of use.

Nine pBAD vectors are currently available: pBAD102/D-TOPO®,pBAD202/D-TOPO®, pBAD-TOPO®, pBAD/Thio-TOPO®, pBAD/His, pBAD/Myc-His,pBAD-DEST49, pBAD/gIII and pBAD/Thio-E. with the following features inall pBAD vectors:

-   -   1. araBAD promoter for dose-dependent regulation    -   2. araC gene for tight control of the araBAD promoter    -   3. Optimized ribosome binding site for increased translation        efficiency    -   4. rrnB transcription termination region for efficient        transcript

The inducible promoters include but are not limited to heat shockpromoters or heavy metal inducible promoters.

This invention provides a process for production of recombinantsuper-compound interferon (rSIFN-co) comprising introducing anartificial gene with selected codon preference into an appropriate host,culturing said introduced host in an appropriate condition for theexpression of said compound interferon and harvesting the expressedcompound interferon.

The process may comprise extraction of super recombinant super-compoundinterferon (rSIFN-co) from fermentation broth, collection of inclusionbodies, denaturation and renaturation of the harvested protein.

The process may maintain the high efficacy even when the recombinantsuper-compound interferon (rSIFN-co) is used with an agent and in aparticular concentration. The process also comprises separation andpurification of the recombinant super-compound interferon (rSIFN-co).The process further comprises lyophilization of the purified recombinantsuper-compound interferon (rSIFN-co). The process comprises productionof liquid injection of recombinant super-compound interferon (rSIFN-co).

In one embodiment, recombinant super-compound interferon (rSIFN-co) wasproduced with recombinant techniques. On the condition of fixed aminoacid sequence, the IFN DNA was redesigned according to the E. Coli.codon usage and then the rSIFN-co gene was artificially synthesized.rSIFN-co cDNA was cloned into the high-expression vector of E. Coli. byDNA recombinant techniques, and a high expression of rSIFN-co was gainedby using of induce/activate-mechanism of L-arabinose to activate thetranscription of P_(BAD) promoter.

Compared with usual thermo-induction, pH induction and IPTG inductionsystems of genetic engineering, arabinose induction/activation systemhas some advantages: (1) Common systems relieve promoter function bycreating a “derepression” pattern. Promoters then induce downstream geneexpression. Temperature and pH change and the addition of IPTG cannotactivate promoters directly. In the system disclosed herein, L-arabinosenot only deactivates and represses but also activates the transcriptionof P_(BAD) promoter which induces a high expression of rSIFN-co.Therefore, the arabinose induction/activation system is a more effectiveexpression system. (2) The relationship between Exogenous andL-arabinose dosage is linear. This means the concentration of arabinosecan be changed to adjust the expression level of the exogenous gene.Therefore, it is easier to control the exogenous gene expression levelin E. Coli. by arabinose than by changing temperature and pH value. Thischaracteristic is significant for the formation of inclusion bodies. (3)L-arabinose is resourceful, cheap and safe, which, on the contrary, arethe disadvantages of other inducers such as IPTG.

This embodiment creates an effective and resistant rSIFN-co-expressingE. Coli. engineering strain with an L-arabinose induction/activationsystem. The strain is cultivated and fermented under suitable conditionsto harvest the bacterial bodies. Inclusion bodies are then purifiedafter destroying bacteria and washing repeatedly. The end result, massof high-purity, spatial-configuration-changed rSIFN-co protein for thisinvention and for clinical treatment, was gained from denaturation andrenaturation of inclusion bodies and a series of purification steps.Said purification would not effect the biological activity of thepurified protein.

The above-described recombinant super-compound interferon (rSIFN-co)possesses anti-viral or anti-tumor activity, and; therefore, is usefulin inhibiting, preventing and treating viral diseases, inhibiting ortreating tumors, or cancers.

Viral Diseases

This invention provides a method for treating or preventing viraldiseases or tumors in a subject comprising administering to the subjectan effective amount of the recombinant super-compound interferon(rSIFN-co) or its equivalent.

As used herein, viral diseases include, but are not limited to,hepatitis A, hepatitis B, hepatitis C, other types of hepatitis,infections caused by Epstein-Barr virus, Human Immunodeficiency Virus(HIV), Ebola virus, Severe Acute Respiratory Syndrome Virus (SARS),Influenza virus, Cytomegalovirus, herpes simplex viruses, other herpesviruses, papovaviruses, poxviruses, picornaviruses, adenoviruses,rhinoviruses, human T-cell leukemia virus I, human T-cell leukemia virusII, or human T-cell leukemia virus III.

In an embodiment, the effective amount is at nanogram level. In anotherembodiment, the virus is Human Immunodeficiency Virus and the effectiveamount is as low as 4 nanograms per milliliter. In another embodiment,the virus is Influenza and the effective amount is as low as 10 nanogramper milliliter.

Inhibition of DNA Replication and Secretion of HBsAg and HBeAg ofHepatitis B Virus.

The recombinant super-compound interferon (rSIFN-co) inhibits the DNAduplication and secretion of HBsAg and HBeAg of Hepatitis B Virus.

Severe Acute Respiratory Syndrome Virus (SARS)

This invention provides a method for preventing or treating Severe AcuteRespiratory Syndrome, or virus-induced upper respiratory diseases, of asubject comprising administering to the subject an effective amount ofrecombinant super-compound interferon (rSIFN-co) or a functionalequivalent thereof. In an embodiment of the above method, the interferonis α, β, γ, ω or a combination thereof.

The recombinant super-compound interferon (rSIFN-co) may be administeredorally, via vein injection, muscle injection, peritoneal injection,subcutaneous injection, nasal or mucosal administration, or byinhalation via a spray or a respirator. In an embodiment rSIFN-co isadministered subcutaneously or intramuscularly at a dose of higher thanor equal to 10 Million International Unit per square meter of surfacearea. In another embodiment rSIFN-co is administered subcutaneously orintramuscularly at a dose of higher than or equal to 20 MillionInternational Unit per square meter of surface area. In an embodiment,the interferon is delivered by a spray device. In a specific embodiment,the device is described in FIG. 11. In one of the embodiments, theinterferon is lyophilized.

This invention provides a method for inhibiting the causative agent ofSevere Acute Respiratory Syndrome, or virus-induced upper respiratorydiseases, comprising contacting the agent with an effective amount ofrecombinant super-compound interferon (rSIFN-co) or its equivalent.

It is determined that the causative agent of SARS is a virus. See eg.Rota et al (2003), Characterization of a Novel Coronavirus Associatedwith Severe Acute Respiratory Syndrome. Science 1085952 and Marra, etal. (2003), The Genome Sequence of the SARS-Associated Coronavirus.Science 1085853.

This invention also provides a method for inhibiting Severe AcuteRespiratory Syndrome virus or Severe Acute Respiratory Syndromevirus-infected cells, or virus-induced upper respiratory diseases, orcells infected with viruses capable of inducing upper respiratorydiseases, comprising contacting an effective amount of the recombinantsuper-compound interferon (rSIFN-co) with said virus or cell. Thiscontact could be direct or indirect.

This invention provides a composition comprising an effective amount ofthe recombinant super-compound interferon (rSIFN-co) capable ofinhibiting Severe Acute Respiratory Syndrome virus or Severe AcuteRespiratory Syndrome virus-infected cells, or virus-induced upperrespiratory diseases, or cells infected with viruses capable of inducingupper respiratory diseases, and a suitable carrier.

This invention provides a composition comprising an effective amount ofthe super-compound interferon capable of preventing or treating SevereAcute Respiratory Syndrome, or virus-induced upper respiratory diseases,of a subject and a suitable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the recombinant super-compound interferon (rSIFN-co)capable of inhibiting Severe Acute Respiratory Syndrome virus or SevereAcute Respiratory Syndrome virus-infected cells, or virus-induced upperrespiratory diseases, and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the recombinant super-compound interferon (rSIFN-co)capable of preventing or treating Severe Acute Respiratory Syndrome, orvirus-induced upper respiratory diseases, in a subject and apharmaceutically acceptable carrier.

This invention provides a device to deliver the above-describedpharmaceutical composition.

In a preferred embodiment, the subject is a human. As it can easily beappreciated, the super-compound interferon can be used in other animalsor mammals.

This invention provides a method for preventing Severe Acute RespiratorySyndrome or virus-induced upper respiratory diseases, in humanscomprising application of the super-compound interferon three times aday via a spray which contains twenty micrograms of interferon, equal toten million units of activity in three milliliter.

Viral Upper Respiratory Infection (VURI)

Viral upper respiratory infection, alternative names common cold, colds.This is a contagious viral infection of the upper respiratory tractcharacterized by inflammation of the mucous membranes, sneezing, and asore throat. It is usually caused by over 200 different viruses, knownas rhinoviruses. Colds are not caused by the same viruses responsiblefor Influenza. Colds are spread through droplets from the coughing orsneezing of others with a cold or by hand contact with objectscontaminated by someone with a cold. The incidence of colds is highestamong children, and the incidence decreases with age because immunity tothe virus causing the cold occurs after the illness. Gradually, immunityto a wide variety of viruses that cause colds is developed in adults.Children may have 10 colds a year, and adults may have 3 colds a year.

The U.S. Centers for Disease Control and Prevention have estimated thatthe average annual incidence of upper respiratory tract infections(URIs) in the United States is 429 million episodes, resulting in morethan $2.5 billion in direct and indirect healthcare costs. The commoncold is most often caused by one of several hundred rhinoviruses (52%),but coronaviruses (8%) or the respiratory syncytial virus (7%) may alsolead to infection. Other viruses, such as influenza (6%), parainfluenza,and adenoviruses, may produce respiratory symptoms, but these are oftenassociated with pneumonia, fever, or chills.

Colds occur in a seasonal pattern that usually begins in mid-Septemberand concludes in late April to early May. The common cold is quitecontagious and can be transmitted by either person-to-person contact orairborne droplets. Upper respiratory symptoms usually begin 1 to 2 daysafter exposure and generally last 1 to 2 weeks, even though viralshedding and contagion can continue for 2 to 3 more weeks. Symptoms maypersist with the occurrence of complications such as sinusitis or lowerrespiratory involvement such as bronchitis or pneumonia.

The common cold has a variety of overt symptoms, including malaise,nasal stuffiness, rhinorrhea, nonproductive cough, mild sore throat,and, in some cases, a low-grade fever. Because of the similarity ofsymptoms, a cold may be mistaken for perennial allergic rhinitis, butallergies can usually be ruled out because of the differences inchronicity.

If a patient presents with a viral URI, the spectrum of remedies isextensive. Since most of these infections are self-limiting, cliniciansusually recommend rest and fluids, but other treatments includeenvironmental and nutritional therapies, over-the-counter andprescription decongestant and antihistamine products, new antihistamineand anticholinergic nasal formulations, and antibiotics. Table 1 listscommonly used cough and cold medications and their side effects.

TABLE 1 A Profile of Common Cough and Cold Medications and Their SideEffects Side Effects and Special Medication Purpose ConsiderationsAerosolized beta2 Reverse Raises heart rate and may agonists (eg,postinflammatory cause tremor albuterol) bronchospasm Alcohol-basedTreat multiple Potential drowsiness and liquid combination symptomscoordination problems products Alpha1 agonists Decongestion May causetachycardia, (oral) (eg, nervousness, transient pseudoephedrine,stimulation, dizziness, phenyl- drowsiness, elevation of propanolamine)blood pressure Anticholinergic Drying May cause nasal dryness andcompounds: occasional epistaxis Ipratropium bromide (topical) OtherDrying May cause orthostasis, anticholinergics dysfunction of heat (eg,regulation, dry mouth, methscopolamine, constipation atropine,hyoscyamine) Antihistamines Drying Drowsiness, dry mouth, (oral) (eg,orthostatic hypertension chlorpheniramine, diphenhydramine) BenzonatateCough suppression, Chewing can numb the capsules local anesthesia mouth;can cause sedation, dizziness Codeine, Cough suppression Drowsiness,constipation, hydrocodone nausea Dextromethorphan Cough suppressionDrowsiness possible, but side effects uncommon Guaifenesin Promote Noside effects; must be expectoration taken with lots of water to(mucolysis) improve efficacy Topical Decongestion Local burning;prolonged use decongestants (eg, may cause dependence oxymetazoline,phenylephrine) Zinc and vitamin C Possible reduction Possible tastedisturbance, lozenges in symptom severity increase of oxalate stones andduration if susceptible

Prevention and Treatment of Upper Respiratory Tract Infections (URI)

Nearly 70˜80% URI are caused by viruses such as respiratory Syncyticalvirus, adenovirus, rhinovirous, cox-sackie virus, corona virus and itsvariant, influenza A virus and its variant, influenza B virus and itsvariant, parainfluenza virus and its variant, or enterovirus and itsvariant. A main cause of URI in adults is from rhinovirous. Forchildren, respiratory syncytical virus and parainfluenza virus are twoleading causes of URI.

Recombinant super-compound interferon (rSIFN-co) plays an important rolein the fight against virus that causes URI. Super-compound interferongains its anti-virus affects mainly via two mechanisms:

-   -   1. Attach to surface of sensitive cells and induce them to        produce anti-virus protein, then block the duplication and        reproduction of viruses in vivo.    -   2. recombinant super-compound interferon (rSIFN-co) can adjust        immune response, including T-cell immune response, activity of        NK cell, the phagocytosis function of monokaryon, and even        formation of some antibodies in vivo.

In treatment for URI, recombinant super-compound interferon (rSIFN-co)can be directly applied to the affected area via a spray or arespiration. This method of treatment allows the interferon to reach thetarget cells first hand. Consequently, marketing the supply as a spray,rather than via oral or injection, would be safer and more effective foradministrating the interferon.

Prevention and Treatment of SARS

With the consent of the Sichuan (a province in China) working group onSARS prevention and control, the distribution of recombinantsuper-compound interferon (rSIFN-co) began in May of 2003.Super-compound interferon spray was allocated to doctors and nurses inhospitals, populated areas with a high risk for SARS, and to theNational research group on prevention and control of SARS. Among the3,000 users as of Dec. 19, 2003, there were no reports of any sideeffects connected to the use of the spray. Furthermore, none of thedoctors and nurses, the people of Sichuan Province, or otherorganizations that have used the Super-compound interferon spray hasbeen infected by SARS.

Therefore, this invention provides a method for inhibiting, preventingor treating virus replication or virus-infected cells by contacting saidvirus or infected cells with an effective amount of the recombinantsuper-compound interferon (rSIFN-co) or its equivalent.

Prevention and Treatment of Tumors

This recombinant super-compound interferon (rSIFN-co) is useful ininhibiting, preventing or treating the following cancers or tumors:

Cancer Skin Cancer Basal Cell Carcinoma Malignant Melanoma Renal cellcarcinoma Liver Cancer Thyroid Cancer Rhinopharyngeal Cancer SolidCarcinoma Prostate Cancer Stomach/Abdominal Cancer Esophageal CancerRectal Cancer Pancreatic Cancer Breast Cancer Ovarian Cancer &Superficial Bladder Cancer Hemangioma Epidermoid Carcinoma CervicalCancer Non-small Cell Lung Cancer Small Cell Lung Cancer GliomaMalignant Leucocythemia Acute Leucocythemia Hemal Chronic LeucocythemiaDisease Chronic Myelocytic Leukemia Hairy Cell Leukemia LymphadenomaMultiple Myeloma Polycythemia Vera Others Kaposi's Sarcoma

Accordingly, this invention provides a method for inhibiting tumor orcancer cell growth by contacting the recombinant super-compoundinterferon (rSIFN-co) or its equivalent with said tumor or cancer cells.

Formulation and Route of Administration

This invention also provides the produced super-compound interferon bythe above processes.

This invention provides a composition comprising recombinantsuper-compound interferon (rSIFN-co) or its equivalent and a suitablecarrier.

This invention provides a pharmaceutical composition comprising therecombinant super-compound interferon (rSIFN-co) or its equivalent and apharmaceutically acceptable carrier.

This invention provides the above-described method wherein recombinantsuper-compound interferon (rSIFN-co) was administered via orally viavein injection, muscle injection, peritoneal injection, subcutaneousinjection, nasal or mucosal administration, or by inhalation via a sprayor a respirator.

This invention provides the above-described method wherein recombinantsuper-compound interferon (rSIFN-co) was administered following theprotocol of injections of 9 μg, 15 μg or 24 μg every two days, 3 times aweek, for 24 weeks.

It was surprising to find that recombinant super-compound interferon(rSIFN-co), the spatial structure of which has been changed, is not onlya preparation to inhibit the DNA duplication of hepatitis B, but toinhibit the secretion of HBsAg and HBeAg on 2.2.15 cells.

One objective of this invention is to offer a preparation of recombinantsuper-compound interferon (rSIFN-co) to directly inhibit the DNAduplication of hepatitis B viruses and the secretion of HBeAg and HBsAgof hepatitis B and decrease them to normal levels.

Formulation

The following are some rSIFN-co preparations: tablets, capsules, liquidsfor oral consumption, pastes, injections, sprays, suppositories, andsolutions. Injections are recommended. It is common to subcutaneouslyinject or vein-inject the medicine. The medicine carrier could be anyacceptable medicine carrier, including carbohydrates, cellulosum,adhesive, collapse, emollient, filling, add-dissolving agent,amortization, preservative, thickening agent, matching, etc.

This invention also provides a pharmaceutical composition comprising theabove composition and a pharmaceutically acceptable carrier.

For the purposes of this invention, “pharmaceutically acceptablecarriers” means any of the standard pharmaceutical carriers. Examples ofsuitable carriers are well known in the art and may include, but are notlimited to, any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution and various wetting agents. Othercarriers may include additives used in tablets, granules, capsules, etc.Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gum, glycols or otherknown excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well-known conventional methods.

Increase of the Half-Life of rSIFN-co

Pegylation

Pegylation is the process by which polyethylene glycol chains areattached to protein and peptide drugs to increase pharmacokinetics byshielding these proteins and peptide drugs from proteolytic enzymes. SeeHarris and Chess, Effect of pegylation on pharmaceuticals. Nat Rev DrugDiscov. 2003 March; 2(3):214-21.

Pegylations is a well-established method for increasing the circulatinghalf-life of protein and liposomal pharmaceuticals based on largehydrodynamic volume of polyethylene glycols. These polyethylene glycolsshield the proteins and peptide drugs from renal clearance, enzymaticdegradation and immune system recognition, thus their half-life andmaking them more acceptable to patients. See Molineux, Pegylation:engineering improved pharmaceuticals for enhanced therapy. Cancer TreatRev. 2002 April; 28 Suppl A: 13-6. The author concludes that pegylationhas beneficial effect on the quality of life of cancer patients.

Pegylation of the interferon increases the amount of time the interferonremains in the body by increasing the size of the interferon molecule bydecreasing the rate of absorption, prolonging the half-life and the rateof interferon clearance. Thus, the duration of biological activity isincreased with pegylated interferon over nonpegylated interferon, thusproviding an advantage over nonpegylated interferons with less frequentadministration and comparable tolerability. The author states thatmonotherapy with pegylated interferon produces a better response in somepatients than monotherapy with the nonpegylated formulation. See Baker,Pegylated Interferons. Rev Gastroenterol Disord. 2001; 1(2):87-99.

Sustained Release or Controlled Release

Sustained release delivery matrices and liposomes maybe used withrSIFN-co to create sustained release and controlled release formulation.See Robinson and Talmadge, Sustained Release of Growth Factors. In Vivo2002 November-December; 16(6): 535-40. The authors state that bothpegylation and sustained release delivery matrices and liposomes improvethe pharmacokinetic and pharmacodynamic properties of recombinantmolecules, and thus by improving clinical efficacy these approachesincrease patient compliance.

This invention provides recombinant super-compound interferon (rSIFN-co)comprising an agent or encapsulated by an agent, capable of affectingthe half-life or delivery of said interferon. In an embodiment thisagent is polyethylene glycol (PEG).

This invention further provides a method for treating or preventingviral diseases or tumors in a subject comprising administering to thesubject an effective amount of the recombinant super-compound interferon(rSIFN-co) or its equivalent comprising an agent or encapsulated by anagent, capable of affecting the half-life or delivery of saidinterferon. In an embodiment this agent is polyethylene glycol (PEG).

This invention will be better understood from the examples which follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS

IFN-con is a new interferon molecule constructed according toconservative amino acids in human IFN-α subtype using geneticengineering methods. It has been proven that IFN-con has broad-spectrumIFN activity, such as high antivirus and tumor inhibition activity,especially for effectively treating hepatitis C.

E. Coli. codon was used to redesign rSIFN-co cDNA and then artificiallysynthesize cDNA of rSIFN-co from published Infergen® (interferonalfacon-1) DNA sequences and deduced amino acid sequences (FIG. 1).

In order to get pure rSIFN-co protein, rSIFN-co cDNA was cloned into E.Coli. high-expression vector, and L-arabinose, which can activate strongP_(BAD) promoter in vectors, was used to induce high expression ofrSlFN-co gene.

Example 1 Synthesis of E. Coli. cDNA Sequence

Redesign of rSIFN-co cDNA Sequence

rSIFN-co cDNA was redesigned according to the codon usage of E. Coli. toachieve high expression in E. Coli. Deduced amino acid sequence from theredesigned cDNA sequence of rSIFN-co is completely coincidental withprimitive amino acid sequence of published Infergen® (interferonalfacon-1) (FIG. 1).

rSIFN-co cDNA Sequence SynthesisrSIFN-co cDNA 5′-Terminus and 3′-Terminus Semi-Molecular Synthesis

Two semi-moleculars can be directly synthesized: rSIFN-co cDNA5′-terminus 280 bp (fragment I) and 3′-terminus 268 bp (fragment II) byPCR. There are 41 bp overlapping among fragment II and fragment I.

(1) Chemical Synthesis Oligodeoxynucleotide Fragment:

Oligomer A: (SEQ ID NO: 10)5′ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGACCGTCACGAC3′ Oligomer B:(SEQ ID NO: 11)5′CTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAAGAATTCGACGGTAACCAGTTCCAGAAAGCTCAGGCTATCTCCGTTCTGCACGAAATGATCCAGCAGACCTTC3′ Oligomer C: (SEQ ID NO: 12)5′GCTGCTGGTACAGTTCGGTGTAGAATTTTTCCAGCAGGGATTCGTCCCAAGCAGCGGAGGAGTCTTTGGTGGAGAACAGGTTGAAGGTCTGCTGGATCATTTC3′ Oligomer D: (SEQ ID NO: 13)5′ATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGATGAAC3′ Oligomer E: (SEQ ID NO: 14)5′GAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTGCGCTTGGG3′ Oligomer F:(SEQ ID NO: 15)5′TTATTCTTTACGACGCAGACGTTCCTGCAGGTTGGTGGACAGGGAGAAGGAACGCATGATTTCAGCACGAACAACTTCCCAAGCGCACGGGGAGTATTTTTTTTCGGTCAGG3′

PCR I for Fragment I: oligodeoxynucleotide B as template,oligodeoxynucleotide A and C as primers, synthesized 280 bp Fragment I.

PCR I mixture (units: μl) sterilized distilled water 39 10xPfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 Oligomer A primer (25 μmol/L) 1 Oligomer C primer (25 μmol/L) 1Oligomer B template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen AmericanLtd.) (25 U/μl) 1 Total volume 50 μl

-   -   PCR Cycle:    -   95° C.2m→(95° C.45s→65° C.1m→72° C.1m)×25 cycle→72° C.10m→4° C.    -   PCR II for Fragment II: oligodeoxynucleotide E as template,        oligodeoxynucleotide D and F as primers, synthesized 268 bp        Fragment II.

PCR II mixture (units: μl) sterilized distilled water 39 10xPfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 Oligomer D primer (25 μmol/L) 1 Oligomer F primer (25 μmol/L) 1Oligomer E template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen AmericanLtd.) (25 U/μl) 1 Total volume 50 μl

-   -   PCR cycle: the same as PCR I        Assembling of rSIFN-co cDNA

Fragment I and II were assembled together to get the complete cDNAmolecular sequence of rSIFN-co using the overlapping and extending PCRmethod. Restriction enzyme Nde I and Pst I were introduced to clonerSIFN-co cDNA sequence into plasmid.

(1) Chemical Synthesis Primers

Oligomer G: (SEQ ID NO: 16) 5′ATCGGCCATATGTGCGACCTGCCGCAGACCC3′Oligomer H: (SEQ ID NO: 17) 5′ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3′

(2) Overlapping and Extending PCR

PCR mixture (units: μl) sterilized distilled water 38 10xPfu buffer(Stratagen American Ltd.)  5 dNTP mixture (dNTP concentration 2.5mmol/L)  2 primer G (25 μmol/L)  1 primer H (25 μmol/L)  1 *fragment Ipreduction (1 μmol/L)  1 *fragment II preduction (1 μmol/L)  1 Pfu DNApolymerase (Stratagen American Ltd.) (2.5 U/μl)  1 Total volume 50μ*Separate and purify PCR production with StrataPrep PCR purification kitproduced by Stratagen American Ltd. And dissolve into sterilizeddistilled water.

-   -   PCR cycle: the same as PCR I        rSIFN-co Gene Clone and Sequence Analysis

pLac T7 plasmid as cloning vector. pLac T7 plasmid is reconstructed withpBluescript II KS(+) plasmid produced by Stratagen (FIG. 3).

Purified PCR production of rSIFN-co cDNA with StrataPrep PCRpurification kit. Digest cDNA and pLac T7 plasmid with NdeI and PstI.Run 1% agarose gel electrophoresis and separate these double-digestedDNA fragments. Recover 507 bp long rSIFN-co DNA fragment and 2.9 kbplasmid DNA fragment. Ligate these fragments by T4 DNA ligase to form arecombinant plasmid. Transform DH_(5α)competent cells (Gibco) with therecombinant plasmid, culture at 37° C. overnight. Identify the positiverecombinant colony, named pHY-1.

Run DNA sequencing with SequiTherm™ Cycle Sequencing Kit produced byAmerican Epicentre Technologies Ltd using L1-COR Model 4000L. Primersare T7 and T3 common sequence primer, the DNA sequencing result matchestheoretic design.

Purify the rSIFN-co, sequence the N-terminus amino acids, the N-terminusamino acid sequence matches experimental design which is as follows:N-Cys-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn-Arg-Arg-Ala-Leu-

Construction, Transformation, Identification, and Hereditary Stabilityof Expression Vector Construction and Transformation of ExpressionVector

Digested E. Coli. expression vector pHY-4 (see FIG. 3) with Nde I tolinearize and subsequently digest with Xba I. Run 1% agarose gelelectrophoresis, and purify the 4.8 kb pHY-4 Nde I-Xba I digest fragmentwith QIAEX II kit produced by QIAGEN Germany Ltd.

At the same time, the pHY-4 plasmid is double digested with Nde I-Xba I.Run 1% agarose gel electrophoresis and purify the 715 bp fragment.Ligate the rSIFN-co and pHY-4 fragments with T4 DNA ligase to constructthe recombinant plasmid (See FIG. 4). Transform DH_(5α)competent cellswith the recombinant plasmid. Spread the transformed cells on LB platewith Amp, 37° C. culture overnight.

Positive Cloning Strain Screening

Randomly choose E. Coli. colonies from above LB-plate, screening thepositive strains containing recombinant vector by endonuclease digestingand PCR analysis. Name one of the positive recombinant plasmid pHY-5,and name the strain containing pHY-5 plasmid PVIII. Amplify and storethe positive strain with glycerol in −80° C.

High Expression of rSIFN-co gene in E. Coli. In pHY-5 plasmid, rSIFN-cogene is under the control of strong promoter P_(BAD). This promoter ispositively and negatively regulated by the product of the gene araC.AraC is a transcriptional regulator that forms a complex with arabinose.In the absence of arabinose, the AraC dimer binds O₂ and I₁, forming a210 bp loop. This conformation leads to a complete inhibition oftranscription. In the presence of arabinose, the dimer is released fromO₂ and binds I₁ and I₂ leading to transcription. Arabinose bindingdeactivates, represses, and even activates the transcription of P_(BAD)promoter, which stimulates P_(BAD), inducing high expression ofrSIFN-co. rSIFN-co expression level in PVIII is more than 50% of thetotal E. Coli. protein.

Summary

rSIFN-CO is a new interferon molecule artificially built according tothe conservative amino acid of human a interferons. It has been provenas an effective anti-hepatitis drug. In order to get enough purerSIFN-co protein, a stable recombinant E. Coli. strain which highlyexpresses rSIFN-co protein was constructed.

First, according to published Infergen® (interferon alfacon-1) aminoacid sequence, E. Coli. codon was used to synthesize the whole cDNA ofrSIFN-co. This DNA fragment was sequenced, proving that the 501 bp codonsequence and TAA termination codon sequence are valid and identical totheocratic design. Subsequent analysis revealed that the N-terminusamino acid sequence and amino acid composed of rSIFN-co produced by therecombinant strain were both identical to the prediction.

The rSIFN-co cDNA was cloned into E. Coli. high-expression vector pHY-4plasmid to construct the recombinant plasmid pHY-5. E. Coli. LMG194strain was further transformed with pHY-4 plasmid to get stable rSIFN-cohigh-expression transformant. This transformant was cultured for 30generations. The heredity of pHY-5 recombinant plasmid in E. Coli.LMG194 was normal and stable, and the expression of rSIFN-co was highand steady.

E. Coli. LMG194, which contains recombinant pHY-5 plasmid, is actuallyan ideal high-expression engineering strain.

REFERENCES

-   1. Blatt L M, Davis J M, Klein S B. et al. The biologic activity and    molecular characterization of a novel synthetic interferon-alpha    species, consensus interferon. Journal of Interferon and Cytokine    Research, 1996; 16(7):489-499.-   2. Alton, K. et al: Production characterization and biological    effects of recombinant DNA derived human IFN-α and IFN-γ analogs.    In: De Maeger E, Schellekens H. eds. The Biology of Interferon    System. 2nd ed. Amsterdam: Elsevier Science Publishers, 1983:    119-128-   3. Pfeffer L M. Biologic activity of natural and synthetic type 1    interferons. Seminars in Oncology, 1997; 24 (3 suppl 9):S9-63—S9-69.-   4. Ozes O N, Reiter Z, Klein S, et al. A comparison of    interferon-con1 with natural recombinant interferons-α antiviral,    antiproliferative, and natural killer-inducing activities. J.    Interferon Res., 1992; 12:55-59.-   5. Heathcote E J L, Keeffe E B, Lee S S, et al. Re-treatment of    chronic hepatitis C with consensus interferon. Hepatology, 1998;    27(4):1136-1143.-   6. Klein M L, Bartley T D, Lai P H, et al. Structural    characterization of recombinant consensus interferon-alpha. Journal    of Chromatography, 1988; 454:205-215.-   7. The Wisconsin Package, by Genetics Computer Group, Inc. Copyright    1992, Medison, Wis., USA-   8. Nishimura, A et al: A rapid and highly efficient method for    preparation of competent E. coli cells. Nuclei. Acids Res. 1990,    18:6169-   9. All molecular cloning techniques used are from: Sambrook,    J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A laboratory    manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbour, N.Y.    1989.-   10. Guzman, L. M et al: Tight regulation, modulation, and high-level    express-ion by vectors containing the arabinose P_(BAD) promoter. J.    Bacteriol. 1995, 177: 4121-4130.    rSIFN-co cDNA Sequence Designed According to E. coli. Codon Usage    and deduced rSIFN-co amino acid sequence

5′   11    21    31   41    51  +1M C D L P Q T H S L G N R R A L I L L A   1ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCTTACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA 5′  71    81    91   101    111  +1Q M R R I S P F S C L K D R H D F G F P  61CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCGGTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC 5′  131  141   151   161   171  +1 Q E E F D G N Q F Q K A Q A I S V L H E121 CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGT TCTGCACGAAGTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCC GATAGAGGCA AGACGTGCTT 5′  191   201   211   221   231  +1M I Q Q T F N L F S T K D S S A A W D E 181ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGC TTGGGACGAATACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACG AACCCTGCTT 5′  251   261   271   281   291  +1S L L E K F Y T E L Y Q Q L N D L E A C 241TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCT GGAAGCTTGCAGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG ACTTGCTGGA CCTTCGAACG 5′     311    321     331   341   351  +1V I Q E V G V E E T P L M N V D S I L A 301GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGA ACGTTGACTC CATCCTGGCTCAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACT TGCAACTGAG GTAGGACCGA 5′     371   381    391   401   411  +1V K K Y F Q R I T L Y L T E K K Y S P C 361GTTAAAAAAT ACTTCCAGCG TATCACCCTG TACCTGACCG AAAAAAAATA CTCCCCGTGCCAATTTTTTA TGAAGGTCGC ATAGTGGGAC ATGGACTGGC TTTTTTTTAT GAGGGGCACG 5′     431   441    451   461   471  +1A W E V V R A E I M R S F S L S I N L Q 421GCTTGGGAAG TTGTTCGTGC TGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAGCGAACCCTTC AACAAGCACG ACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC 5′   491   501  +1 E R L R R K E #            (SEQ ID NO: 1) 481GAACGTCTGC GTCGTAAAGA ATAA (SEQ ID NO: 2)CTTGCAGACG CAGCATTTCT TATT (SEQ ID NO: 3)

Example 2 Separation and Purification of rSIFN-co 1. Fermentation

Inoculate the recombinant strain in LB media, shaking (200 rpm) under37° C. overnight (approximate 18 h), then add 30% glycerol to thefermentation broth to get final concentration of 15%, allotted to 1 mltube and kept in −20° C. as seed for production.

Add 1% of the seed to LB media, shaking (200 rpm) under 37° C. overnightto enlarge the scale of the seed, then add to RM media with a ratio of10%, culturing under 37° C. Add arabinose (20% solution) to 0.02% as aninductor when the OD₆₀₀ reaches about 2.0. 4 hours after that, stop theculture process, collect the bacteria by centrifuge, resuspend thepellet with buffer A, and keep in −20° C. overnight. Thaw and break thebacteria by homogenizer, then centrifuge. Wash the pellet with buffer B,buffer C, and distilled water to get a relatively pure inclusion bodies.

2. Denaturation and Renaturation

Dissolve the inclusion body in Guanidine-HCl (or urea) of 6 mol/L. Thesolution will be a little cloudy. Centrifuge it at a speed of 10000 rpm.Determine the protein concentration of the supernatant. This supernatantis called “denaturation solution.” Add the denaturation solution torenaturation buffer, and keep the final protein concentration under 0.3mg/ml. It is better to add the totally denatured solution in three stepsinstead of one step. Keep the solution overnight under 4° C. Afterwards,dialyze 10 mol/L, 5 mol/L PB buffer and distilled water, then adjust itspH by 2 mol/L HAc-NaAc. Let it stand, then filtrate.

3. Purification

-   -   POROS HS/M anion exchange chromatography:

Condense the eluted solution by POROS HS/M. Sometimes a purification bysephacryl S-100 step can be added to meet stricter purity requirements.

Note:

-   Buffer A: 100 mmol/L Tris-HCl, pH 7.5-10 mmol/L EDTA-100 mmol/L NaCl-   Buffer B: 50 mmol/L Tris-HCl, pH 7.5-1 mol/L Urea-10 mmol/L    EDTA-0.5% Triton X-100-   Buffer C: 50 mmol/L Tris-HCl, pH 7.5-2 mol/L Urea-10 mmol/L    EDTA-0.5% Triton X-100-   Buffer D: 1 mol/L NaCl- - -50 mmol/L Na₂HPO₄ (pH 5.5)-   Buffer E: 1 mol/L NaCl- - -50 mmol/L Na₂HPO₄ (pH 5.0)-   Buffer F: 1 mol/L NaCl- - -50 mmol/L Na₂HPO₄ (pH 4.0)-   Buffer G: 1 mol/L NaCl- - -50 mmol/L Na₂HPO₄ (pH 3.6)-   Renaturation buffer: 0.5 mol/L Arginine—150 mmol/L Tris-HCl, pH    7.5-0.2 mmol/L EDTA

LB Media: 1 L

Tryptone 10 g Yeast extracts  5 g NaCl 10 g

RM Media: 1 L

Casein  20 g MgCl   1 mmol/L (0.203 g) Na₂HPO₄   4 g; KH₂PO₄   3 g, NaCl0.5 g NH₄Cl   1 g

After purification, the buffer was changed to PBS (pH 7.0) along withthe step of condensing by POROS HS/M. This is called the “Protein StockSolution.” It can be directly used in the preparation of injections orsprays, or stored at 2-8° C.

Formula for Injection:

Solution Lyophilized powder Solution of rSIFN-co 34.5 μg/ml 34.5 μg/mlPB (pH7.0)   25 mmol/L   10 mmol/L Glycine —  0.4 mol/L NaCl  0.1 mol/L—

For Spray:

EDTA 0.01% Tween 80 0.05% Trisodium citrate 10 mmol/L Glycerol 1.26%Sodium Chloride 0.03% Phenylmethanol  0.5% HSA  0.1% rSIFN-co 10 μg/ml

Quality Control Process

During purification, tests for protein content, protein purity, specificactivity and pyrogen are conducted after each step. When the stocksolution is obtained, all the tests listed in the table are done oneafter the other.

The quality of the product is controlled according to “ChineseRequirements for Biologics.”

1. Original Protein Solution

Item of Test Method Protein Stock Solution: Test for Protein ContentLowry Test for Protein Purity Non-reductive SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) HPLC Analysis Test forMolecular Weights Reductive SDS-PAGE Test for Specific ActivityAccording to Method in “Specific Activity Test of Interferon Test forLeftover Exogenetic Using DNA Labeling and DNA Detection Kit Test forActivity of According to Method in Leftover Antibiotics “Chemical andOther Test Methods for Biologics” Test for Bacterial Endotoxin Accordingto Method in “Requirements for Bacterial Endotoxin Test of Biologics”Test for Isoelectronic Point Isoelectric Focusing Electrophoresis Testfor Identify UV spectrum (range of Characteristics of the wavelength:190-380 nm) Protein Peptide Mapping (hydrolyzed by pancreatic enzyme,analyzed by C-18 column) N-terminal Sequence Test C-terminal SequenceTest Circular Dichroism Amino Acid Analysis Semi-finished Product Testfor Bacterial Endotoxin According to Method in “Requirements forBacterial Endotoxin Test of Biologics” Product Appearance Check ChemicalAccording to Method in “Chemical and Other Test Methods for Biologics”Test for Specific Activity According to Method in “Specific ActivityTest of Interferon Sterility Test According to Method in “c” AbnormalToxicity Test Test on Mouse Pyrogen Test According to Method in“Requirements for Pyrogen Test of Biologics” Test for Stability ofProduct Note: “Chemical and Other Test Methods for Biologics”,“Requirements for Pyrogen Test of Biologics” and “Requirements forBacterial Endotoxin Test of Biologics” all can be found in the “ChineseRequirements for Biologics.” “Chinese Requirements for Biologics,” PANZhengan, ZHANG Xinhui, DUAN Zhibing, et al. Chinese BiologicsStandardization committee. Published by Chemical Industry PublishingCompany, 2000.

Example 3 Stability of Lyophilized Powder of Recombinant Super-CompoundInterferon Injection

The stability experiments were carried out with samples of lyophilizedpowder of recombinant super-compound interferon (rSIFN-co) injection intwo specifications and three batches. The experiments started in April2000.

1. Sample Source

Samples were supplied by Sichuan Huiyang Life-engineering Ltd., SichuanProvince. Lot: 990101-03, 990101-05, 990102-03, 990102-05, 990103-03,990103-05

2. Sample Specifications

Every sample in this experiment should conform with the requirements inthe table below.

TABLE 1 Standard of Samples in Experiment Items Standards 1. Appearancewhite loose powder 2. Dissolving time dissolve rapidly in injectionwater (within 2 min) at room temperature 3. Clarity colorless liquid orwith little milk-like glisten; should not be cloudy, impurity or withindiscernible deposit 4. pH value 6.5~7.5 5. Potency (IU/dose) 80%~150%of indicated quantity (9 μg: 4.5 × 10⁶ IU, 15 μg: 7.5 × 10⁶ IU) 6.Moisture no more than 3.0% ( W/W)

3. Experimental Content

Test samples at 2˜8° C.: The test samples were put into a 2˜8° C.refrigerator, then the above items of these samples were respectivelytested in the 1^(st), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th),30^(th), 36^(th) month. The results were recorded.

Test samples at 25° C.: The test samples were put into a thermostat at25° C., then the above items of these samples were respectively testedin the 1^(st), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th),30^(th) month. The results were recorded.

Test samples at 37° C.: The test samples were put into a thermostat at37° C., then the above items of these samples were respectively testedin the 1^(st), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th) month.The results were recorded.

4. Results and Conclusion

-   -   1) At 37° C., according to data collected at designated points        during testing and compared with data before testing, the        potency began descending from the 6^(th) month and the changes        in the three batches were similar. The appearance of other items        had no changes.    -   2) At 25° C., according to data collected at designated points        during testing and compared with data before the testing, the        potency only had a little change, and the changes in the three        batches were similar. The appearance of other items had no        changes.    -   3) At 2-8° C., according to data collected at designated points        during testing and compared with data before testing, the        potency of the three batches all were stable. The appearance of        other items also had no changes.    -   In conclusion, it is suggested that the lyophilized powder of        recombinant super-compound interferon for injection should be        better stored and transported at low temperatures. Without such        conditions, the product can also be stored for short periods        (i.e., 3 months) at room temperature.

Example 3.5 Production Flow Chart of rSIFN-co 1. Production

1.1 Fermentation

-   -   Use mixture of LB+M9 as culturing medium. The amount of        innoculum will be 1.5%. Agitate to OD₆₀₀=0.4 (about 3.5 hours)        under 32° C., then raise temperature to 42° C. Continue the        agitation for another 6 hours, the expression of rSIFN-co will        reach the maximum level. The examination under scanning of the        gel resulting from SDS-PAGE shows that the level of expression        is up to 57%, which is the highest standard in China.

1.2 Purification

The purity of the product (rSIFN-co) from this production procedure isshown to 95% under the test of SDS-PAGE where molecular weight is 14.5Kda. The reverse phase HPLC shows a single peak and the purity is up to97%. Its specific activity is up to 1×10⁹ IU/mg protein.

1.3 Packaging and Inspection

-   -   After HPLC purification, 2% human serum albumin, 1% sucrose and        1% glucose are added to the rSIFN-co. It is then separated and        lyophilized into injection sample. When tested under the        Wish-VVS inspection system, the result was 4.5×10⁸ IU. When        tested with aseptic inspection and pyrogen inspection under the        standard requirement of China, the results were negative. This        result complies with the requirements for IV injection.

2. Quality Control

2.1 Biological Characteristics

-   -   (1) When using LB+M9 to cultivate bacteria, the characteristics        should match with the typical characteristics of E-coli        bacteria. No other bacteria were detected.    -   (2) When smeared for Gram staining and inspected under a        microscope, it is bacteria-negative.    -   (3) Reaction to antibiotics is the same as those original        bacteria.    -   (4) Electron microscope inspection shows typical characteristics        of E-coli bacteria. No mycoplasma, virus spore or other micro        pollutes was detected.    -   (5) Biochemical reaction test shows characteristics of E-coli        bacteria.

2.2 Quality Control of Interferon Expression

-   -   (1) Interferon expression (cultivated in an agitating platform)        matches the amount of expression in original input bacteria.    -   (2) When tested with anti-interferon serum, a reaction is shown.    -   (3) Plasmid inspection: Restriction digest matched with the        original plasmid.

2.3 Bacteria Strain Product

-   -   Bacteria strain product denotes the specimen from the original        bacteria strain that was produced from the procedures shown in        1.2.    -   The bacteria strain product should be inspected as follows to        make sure there is no derivation: Use LB to plate 2-3 pieces and        cultivate. Separate and take 5-10 bacteria groups for the test        of interferon expression. Repeat the test at least two (2)        times. Only use the one which shows the highest % to be the        bacteria strain product.

2.4 Inoculum

-   -   The inoculum denotes the chosen bacteria strain product after        fermentation. The amount, cultivation time and most appropriate        OD value of inoculum can be decided according to bacteria        strain. An anti-polluted bacteria procedure should apply for        whatever inoculum would be produced.

2.5 Growing of Bacteria Strain

-   -   Growing of bacteria strain would be done in a Bacteria Free room        environment where no more than one bacterium is growing in the        same room. Same culturing medium will be used for both bacteria        strain and inoculum. The one used in rSIFN-co is LB.

2.6 Fermentation

-   -   (1) Fermentation only takes place in a clean fermentation room        with a single bacteria fermentation environment.    -   (2) Cleaning of fermentation container and tube is done twice,        before and after the insertion of culturing medium. Then, the        container should be frozen to reach the appropriate temperature        for inoculum.    -   (3) Avoid using antibiotic which might affect cell growth in the        culturing medium.    -   (4) Fermentation parameters like temperature, pH value,        dissolved oxygen and time required could be varied according to        different types of bacterial strains.

2.7 Bacteria Collection

-   -   (1) Centrifuge the bacteria solution to collect bacteria or use        another method. All apparatus should be cleaned before and after        the operation. The waste solution should be drained after the        cleaning procedure.    -   (2) The bacteria should be kept under 4-8° C. if they are going        to be split within 24 hours. Otherwise, they should be kept        under −30° C. Those are kept under such conditions can be used        within 6 months.

2.8 Bacteria Cell Lysis

-   -   (1) Use appropriate buffer solution to balance the bacteria        strain. Cell lysis can be done by physical, chemical or        biological methods. Use centrifuge to precipitate the bacteria        and apply cleaning solutions.    -   (2) If the chemical method is used to split cells, no solutions        harmful to human beings should be used.

2.9 Purification

-   -   (1) Purification will get rid of most of the non-interferon        contents. In the process of purification, no toxic materials        should be found if extra elements are added.    -   (2) If using antibody affinity chromatography for purification,        there should be an indication of the source and degree of        purity. Also, inspection of small quality IgG should be        performed.    -   (3) During the process of purification, clearance of pyrogen is        critical. All apparatus should be checked to eliminate this        interference.    -   (4) The highly concentrated interferon is known as “intermediate        product”. After inspection and tests, add albumin to raise the        concentration to 2% which is now known as “albumin intermediate        product”. After examination and tests, it should be kept at        −30° C. and never thawed before use. This product should be used        within 6 months.    -   (5) The albumin that is used in this process should also fulfill        tests and requirements such as: negativity under RBSAG        inspection and an indication of the ratio among monomer, dimer        and polymer.

2.10 Production into Tube Product

-   -   (1) Filtration: Use 0.22μ membrane to filter the bacteria. The        product should be handled with aseptic techniques. Samples        should be taken to test the value of the interferon.    -   (2) Dilution: Dilute the albumin intermediate product with 2%        diluent. No preservative should be added. The product can be        lyophilized after the aseptic inspection and pyrogen inspection.

2.11 Lyophilization

-   -   The lyophilization should not affect the activity of interferon,        and the water content of said lyophilite will be maintained.

2.12 Inspection

-   -   There are two types of rSIFN-co made. One is for injection and        the other for topical use. The specifications for the two are        different. There are intermediate products and final products        for each type. In the injection type, intermediate products        include purified interferon, albumin intermediate product, and        bacteria free albumin intermediate product. Final product from        the injection type will denote only lyophilized product. The        intermediate product in the topical type denotes only purified        interferon. The final product from the topical type denotes only        separated packed liquid formed lyophilized products.

2.13 Packaging

-   -   There is different packaging for the injection type and the        topical type.

2.14 Storage

-   -   The product should be kept at 4° C. The purification solution        should not be stored in a frozen state.

2.15 Expiration

-   -   The expiration period is two (2) years after the lyophilization        procedure for lyophilized products. The expiration period is 6        months after individual packing for liquidated products.

Example 4 Preparation of rSIFN-co

Preparation of lyophilized injection Lyophilized powder Stock Solutionof 34.5 μg/ml rSIFN-co PB (pH7.0) 10 mmol/L Glycine 0.4 mol/L

Preparation technique: Weigh materials according to recipe. Dissolvewith sterile and pyrogen-free water. Filter through 0.22 μm membrane tode-bacterialize, preserve at 6-10° C. Fill in vials after affirming theyare sterile and pyrogen-free, 0.3 ml/vial or 0.5 ml/vial, and lyophilizein freeze dryer.

Preparation of liquid injection Solution Stock Solution of 34.5 μg/mlrSIFN-co PB (pH7.0) 25 mmol/L NaCl 0.1 mol/L

Preparation: Weigh materials according to recipe. Add to desired levelwith sterile and pyrogen-free water. Filter through 0.22 μm membrane tode-bacterialize, preserve at 6-10° C. Fill in airtight vial afteraffirming it is sterile and non-pyrogen at 0.3 ml/vial or 0.5 ml/vial.Store at 2-10° C., and protect from light.

Example 4.5 Acute Toxicity of rSIFN-co

Treat mice with large dose (150 μg/kg, equal to 1000 times of the normaldose per kilo used in treatment of adult patients) of rSIFN-co at onetime by intramuscular injection. Then observe and record their deathsand toxic reactions. Results show that: 24 hours after injection, noabnormal reaction had been recorded. The organs of the animals which hadbeen selected to be killed also had no signs of abnormal changes. Thoseremaining mice were all kept alive and were normal after two weeks. Theweights of mice in the experimental group and control group allincreased, and the ratio of increase showed no obvious differencebetween the two groups (P>0.05) according to their weights on thefourteenth day. No abnormal changes were seen from the main organs ofthose mice after two weeks.

1. Experimental Material 1.1 Animals

40 healthy adult mice, weighing 18-22 g, half male and half female,qualified by Sichuan experiment animal control center.

1.2 Medicines

rSIFN-co (Provided by Sichuan Huiyang Life-engineering Ltd.) sterilizedsolution, 0.15 mg/ml, Lot: 981201

rSIFN-co was administered i.m. in saline.

2. Method

Separate the 40 mice into two groups randomly, one for experimentalmedicine, another for control. Inject medicines or saline at the sameratio (0.1 ml/10 g) through muscle to each mouse according to whichgroup they belong. (150 μg/kg of rSIFN-co for experimental group; andsaline for control group). After injection, observe and record acutetoxicity shown in mice. Kill half of the mice (male and female eachhalf) to check whether there were any abnormal pathologic changes intheir main organs, such as heart, spleen, liver, lung, kidney, adrenalgland, stomach, duodenum, etc. after 24 hours. Those that remain arekept and observed until the fourteenth day. Weigh all mice, kill them,and then observe the appearance of the organs listed above to see ifthere are any abnormalities. Take pathological tissue and examine it,using the examination to assess the difference in weight increases inthe two groups.

3. Results

Results show that there was no acute toxicity seen after all mice weretreated with i.m. rSIFN-co with 150 μg/kg at a time, equal to 1000 timesthe normal dose per kilo used in treatment of adult patients. In the 14days after injection, all mice lived well. They ate, drank, exercised,and excreted normally and showed normal hair conditions. None of themdied. The observation of the main organs of the randomly selected miceshows no abnormal changes 24 hours after injection. 14 days afterinjection, all remaining mice were killed. Autopsies also showed nochanges. The weights of mice in the two groups all increased, but noobvious difference was shown when accessed with statistic method(p>0.05). See Table 6.1:

TABLE 6.1 Influence to weights of mice after injection of rSIFN-coWeights Weights Increased before after value of injection injectionweights Group Dose Animal (g) (g) (g) Control 0 20 19.8 ± 1.7 30.8 ± 2.811.0 ± 2.9 rSIFN-co 150 20 19.4 ± 1.7 32.1 ± 3.3 12.7 ± 4.3

4. Conclusion

Under conditions of this experiment, there were no toxic reactions inall mice after injection of rSIFN-co with 150 μg/kg. The conclusion canbe reached that the maximum tolerable dose of i.m. in mice is 150 μg/kg,which is equal to 1000 times the normal dose per kilo used in treatmentof adult patients.

2002 rSIFN-co Drug Inspection Report:

Nov. 14, 2002 rSIFN-co Drug Inspection Report by China Drugs &Biological Products Inspection Laboratory.

On Nov. 14, 2000, 80 vials of rSIFN-co each containing 9 μg (micrograms)provided by Sichuan Biotechnology Research Center were tested. rSIFN-coDrug was white in color with produced no precipitation when water wasadded. The pH value was 6.9 while the standard was between 6.5 to 7.5.The water content of rSIFN-co was 2.3% while the standard was smallerthan 3.0%. Test for bacteria showed no bacterial grown. rSIFN-co passedpyrogen test. The toxicity test on mice showed no harm. Mice were aliveand gained weight. The specific activity test was 6.0×10⁶ IU/vial whilethe standard was between 3.6×10⁶ IU/vial to 6.8×10⁶ IU/vial. Theidentification test was positive.

Example 5

Crystal Growth of rSIFN-co and Test of Crystallography Parameter

Crystal of rSIFN-co. Two types of crystal were found aftersystematically trial and experiment. (See FIGS. 7-9)

1. Crystal Growth

-   -   Dissolve rSIFN-co protein with pure water (H₂O) to 3 mg/ml in        density. Search of crystallization by using Hampton Research        Crystal Screen I and II which was made by Hampton Company. By        using Drop Suspension Diffusion Method, liquid 500 μl, drop 1 μl        protein+1 μl liquid, in 293K temperature. First 2 different        types of small crystals were found as listed in Table 12.1.

TABLE 12.1 Screen of rSIFN-co Crystallin Condition I II Diluent 0.1MTris-HCl 0.1M HEPES PH = 8.75 PH = 7.13 Precipitant 17.5% (w/v) PEG550MME 10% (w/v) PEG6K Additives 0.1M NaCl 3% (w/v) MPD Temperature 293K293K Crystal Size (mm) 0.2 × 0.2 × 0.1 0.6 × 0.02 × 0.02 CrystallogramFIG. 7 FIG. 8

2. Data Collection and Processing

-   -   Crystal I was used to collect X-Ray diffraction data and        preliminary analysis of crystallography. Parameters were also        tested. The diffraction data was collected under room        temperature. Crystal I (Condition I) was inserted into a thin        siliconized wall tube. Using BrukerAXS Smart CCD detector, the        light source is CuKα (λ=1.5418 Å) generated by Nonius FR591        X-ray generator. Light power 2000 KW (40 kv×50 mA), wave length        1.00 Å, under explosion 60 second, Δφ=2°, the distance between        crystal and detector was 50 mm. Data was processed for using        Proteum Procedure Package by Bruker Company. See FIG. 9 for        crystal diffraction pattern (partially). See Table 12.2 for the        result of the process.

TABLE 12.2 Results of Crystallography Parameters Parameters a (Å)  82.67b (Å) 108.04 c (Å) 135.01 α (Å)  90.00 β (Å)  90.00 γ (Å)  98.35 SpaceGroup P2 or P2₁ Sharpness of separation 5 Å Asymmetric molecule #  10Dissolution  57.6%

Besides, there was no crystal growth of rSIFN-co based on previouspublications. The closest result to the rSIFN-co was huIFN-α2b but thescreen was very complicated. After seeding 3 times, crystal grew to0.5×0.5×0.3 mm, sharpness of separation was 2.9 Å, space group was P2₁.The crystals were also big, asymmetric molecule number was 6, anddissolution was about 60%.

Clinical Report 1:

Evidence of effectiveness of rSIFN-co in healing cancer. See FIGS.17A-H.

The ultra sound inspection showed an enlarged right ovary and abdominalfluid. The patient was suspected of having ovarian cancer.

Western China No. 2 Hospital reported a patient with ovarian cancer andbreast gland cancer diagnosed on Jul. 14, 2004. Her serum containedCA-125>600 U/ml and CA-153>250 U/ml. Also 2000 ml abdominal water wasfound. On Jul. 16, 2004, malignant cancer cells and low differentialgland cancer cells (likely a low graded differential Adenocarcinoma)were found from the abdominal water and cancer cells and death materialswere found from the mammary gland check up. On Aug. 4, 2004, it wasconcluded diagnosis as ovary cancer.

The patient was treated with rSFIN-co starting Jul. 14, 2004. She wasinjected with 15 μg of rSFIN-co on Jul. 14, 2004, Jul. 16, 2004, Jul.18, 2004, Jul. 20, 2004 and Jul. 22, 2004 respectively. She beganchemotherapy on Jul. 22, 2004. On Aug. 3, 2004 abdominal surgery wasperformed. It was expected that her abdominal water would be more than2000 ml. However, only 200 ml were recorded. On Aug. 4, 2004 theexamination results showed she had mammary gland cancer, ovarian cancerof right and left ovary and lymphoma. She was treated with rSIFN-co andchemotherapy at the same time. She did not have operation on mammaryglands.

On Dec. 27, 2004 the examination report showed her CA-125 dropped to 5U/ml and CA-153 dropped to 13 U/ml. On Feb. 25, 2005, her PETexamination report from Daping Hospital, Third Military MedicalUniversity of PRC showed there was no obvious abnormal difference onmetabolic reactions on her body and brain. The symptoms of her mammarygland cancer disappeared. No traces of cancer were found.

PET Imaging:

On Feb. 25, 2005 PET imaging report on Feb. 25, 2005 of this 43 yearsold patient diagnosis with left side ovary cancer and was treated withrSIFN-co since Jul. 14, 2004; PET imaging was done at PET Center of theDaping Hospital, Third Military Medical University of PRC.

Fasting patient was intravenously injected with ¹⁸F-FDG14.8mCi. Brainimages were taken 50 minutes after injection. The images were clear, noobvious abnormal increase or decrease of radiation were observed oncerebral epidermis, both sides of cerebellum, both sides of hypothalamusand basal.

-   -   Whole body imaging was done 60 minutes after injection. The        images were clear. No obvious abnormal increase or decrease of        radiation on neck, lungs, mediastinum, liver, both sides of        adrenals, abdominal lymph gland, pelvic cavity, bones.

The image of heart was clear.

Result: The FDG-PET images of the whole body and brain did not showabnormal FDG metabolic increase or decrease after five-and-half (5.5)months of rSIFN-co treatment of ovarian ovary cancer.

Conclusion: Comparison of CA-153 and CA-125 levels before and afterrSFIN-co treatment evidenced that rSFIN-co is effective against breastand ovarian cancer.

Clinical Report 2:

A kidney cancer patient was treated in the following manner. In ahalf-month period, the patient was given 3 injections of 9 μg ofrSIFN-co and 3 injections of 15 μg of rSIFN-co. In the one and a halfmonths following these injections, he received 24 μg injections ofrSIFN-co every day. A kidney biopsy showed no metastasis after thiscourse of treatment. The patient showed a full recovery. Every half yearafter recovery, the patient received 15 μg injections of rSIFN-co 15times over a one-month period.

Example 6 rSIFN-co Inhibits HBV-DNA Duplication and Secretion of HBsAgand HBeAg

Materials

Solvent and Dispensing Method: Add 1 ml saline into each vial, dissolve,and mix with MEM culture medium at different concentrations. Mix on thespot.

Control drugs: IFN-α2b (Intron A) as lyophilized powder, purchased fromSchering Plough. 3×10⁶ IU each, mix to 3×10⁶ IU/ml with culture medium;Infergen® (liquid solution), purchased from Amgen, 9 μg, 0.3 ml each,equal to 9×10⁶ IU, and mix with 9×10⁶ IU/ml culture medium preserve at4° C.; 2.2.15 cell: 2.2.15 cell line of hepatoma (Hep G2) cloned andtransfected by HBV DNA, constructed by Mount Sinai Medical Center.

Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum,HycloneLab American Ltd. G-418 (Geneticin); MEM dispensing, GibcoAmerican Ltd.; L-Glutamyl, imported and packaged by JING KE ChemicalLtd.; HBsAg and HBeAg solid-phase radioimmunoassay box, NorthwardReagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern ChinaMedicine; And Lipofectin, Gibco American Ltd.

Experimental goods and equipment: culture bottle, Denmark Tunclon™;24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxidehatching box, Shel-Lab American Ltd.; MEM culture medium 100 ml: 10%cattle fetal blood serum, 3% Glutamyl 1%, G418 380 μg/ml, biograncetina50 U/ml.

Method:

2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box withfull of 2.2.15 cell, digest at 37° C. for 3 minutes, and add culturemedium to stop digest and disturb it to disperse the cells, reproducewith ratio of 1:3. They will reach full growth in 10 days.

Toxicity test: Set groups of different concentrations and a controlgroup in which cells are not acted on with medicine. Digest cells, anddispense to a 100,000 cell/ml solution. Inoculate to 96-well cultureboard, 200 μl each well, culture at 37° C. for 24 h with 5% CO₂. Testwhen simple cell layer grows.

Dispense rSIFN-co to 1.8×10⁷ IU/ml solution, then prepare a series ofsolutions diluted at two-fold gradients. Add into 96-well culture board,3 wells per concentration. Change the solution every 4 days. Testcytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as3, 50% as 2, 25% as 1, zero as 0. Calculate average cell lesion andinhibition rate of different concentrations. Calculate TC₅₀ and TC₀according to the Reed Muench method.

${TC}_{50} = {{Antilog}\left( {B + {\frac{50 - B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration,C=log dilution power

Inhibition test for HBeAg and HBsAg: Separate into positive and negativeHBeAg and HBsAg contrast groups, cell contrast group and medicineconcentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into6-well culture board, 3 ml each well, culture at 37° C. for 24 h with 5%CO₂, then prepare 5 gradiently diluted solutions with 3-fold as thegrade (Prepare 5 solutions, each with a different protein concentration.The concentration of Solution 2 is 3 times lower than that of Solution1, the concentration of Solution 3 is 3 times lower than that ofSolution 2, etc.) 4.5×10⁶ IU/ml, 1.5×10⁶ IU/ml, 0.5×10⁶ IU/ml, 0.17×10⁶1U/ml, and 0.056×10⁶1 U/ml, 1 well per concentration, culture at 37° C.for 24 h with 5% CO₂. Change solutions every 4 days using the samesolution. Collect all culture medium on the 8^(th) day. Preserve at −20°C. Repeat test 3 times to estimate HBsAg and HBeAg with solid-phaseradioimmunoassay box (Northward Reagent Institute of Chinese IsotopeLtd.). Estimate cpm value of each well with a γ-accounting machine.

Effects calculation: Calculate cpm mean value of contrast groups anddifferent-concentration groups and their standard deviation, P/N valuesuch as inhibition rate, IC50 and SI.

${\left. 1 \right){Antigen}\mspace{14mu} {inhibition}\mspace{14mu} {{rate}(\%)}} = {\frac{A - B}{A} \times 100}$

-   -   A=cpm of control group; B=cpm of test group;    -   2) Counting the half-efficiency concentration of the medicine

${{Antigen}\mspace{14mu} {inhibition}\mspace{14mu} {IC}_{50}} = {{Antilog}\left( {B + {\frac{50 - B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration,C=log dilution power

-   -   3) SI of interspace-conformation changed rSIFN-co effect on        HBsAg and HBeAg in 2.2.15 cell culture

${SI} = \frac{{TC}_{50}}{{TC}_{50}}$

-   -   4) Estimate the differences in cpm of each dilution degree from        the control group using student t test

Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days.Exsuction culture medium (Separate cells from culture medium by means ofdraining the culture medium). Add lysis buffer to break cells, thenextract 2 times with a mixture of phenol, chloroform and isoamyl alcohol(1:1:1), 10,000 g centrifuge. Collect the supernatant adding anhydrousalcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20 μlTEbuffer. (2) Electrophoresis: Add 6×DNA loading buffer, electrophoresison 1.5% agarose gel, IV/cm, at fixed pressure for 14-18 h. (3)Denaturation and hybridization: respectively dip gel into HCl,denaturaion buffer and neutralization buffer. (4) Transmembrane: Make anorderly transfer of DNA to Hybond-N membrane. Bake, hybridize and exposewith dot blot hybridization. Scan and analyze relative density withgel-pro software. Calculate inhibition rate and IC₅₀.

Results

Results from Tables 4.1, 4.2 and 4.3 show: After maximum innocuousconcentration exponent culturing for 8 days with 2.2.15 cell, the maximais 9.0±0×10⁶ IU/ml average inhibition rate of maximum innocuousconcentration rSIFN-co to HBeAg is 46.0±5.25% (P<O. 001), IC₅₀ is4.54±1.32×10⁶ IU/ml, SI is 3.96; rate to HBsAg is 44.8±6.6%, IC₅₀ is6.49±0.42×10⁶ IU/ml, SI is 2.77. This shows that rSIFN-co cansignificantly inhibit the activity of HBeAg and HBsAg, but that the IFNof the contrast group and Infergen® cannot. It has also been proven inclinic that rSIFN-co can decrease HBeAg and HBsAg or return them tonormal levels.

TABLE 4.1 Results of inhibition rate of rSIFN-co to HBsAg and HBeAgInhibition rate Average 1- Accumulated Concentration First Second ThirdFirst Second Third inhibition Accu- inhibition (×10⁴ IU/ml) well wellwell well well well rate Accumulation mulation rate First batch:(rSIFN-co) Inhibition effect to HBeAg 900 9026 8976 10476 0.4362270.43935 0.345659 0.407079 0.945909 0.592921 0.614693546 300 9616 1208210098 0.3993754 0.245347 0.369269 0.337997 0.5388299 1.2549240.300392321 100 9822 16002 12800 0.386508 0.0005 0.2005 0.1958360.200833 2.059088 0.08867188 33.33333 15770 19306 16824 0.014991 0 00.004997 0.0049969 3.054091 0.001633453 11.11111 19172 22270 18934 0 0 00 0 4.054091 0 Control Cell 16010 Blank 0 Dilution 3 IC50 602.74446016Inhibition effect to HBsAg 900 7706 7240 7114 0.342155 0.381936 0.3926930.372261 0.922258 0.627739 0.595006426 300 8856 7778 9476 0.24398160.336008 0.191053 0.257014 0.5499972 1.370724 0.286349225 100 1081810720 10330 0.07649 0.084856 0.118149 0.093165 0.292983 2.277560.113977019 33.33333 10744 11114 10570 0.082807 0.051221 0.0976610.07723 0.1998179 3.20033 0.058767408 11.11111 10672 9352 10810 0.0889530.201639 0.077173 0.122588 0.122588 4.077742 0.02918541 Control Cell11714 Blank 0 Dilution 3 IC50 641.7736749 Second batch: (rSIFN-co)Inhibition effect to HBeAg 900 7818 8516 9350 0.554378 0.514592 0.4670540.512008 1.371181 0.487992 0.737521972 300 10344 10628 9160 0.41039670.394209 0.477884 0.427497 0.8591731 1.060496 0.447563245 100 1229614228 13262 0.299134 0.18901 0.244072 0.244072 0.4316522 1.8164230.19201839 33.33333 15364 17414 16188 0.124259 0.00741 0.77291 0.0696530.1876045 2.74677 0.063933386 11.11111 17386 13632 15406 0.0090060.222982 0.121865 0.117951 0.117951 3.628819 0.03148073 Control Cell16962 Blank 0 Dilution 3 IC50 365.9357846 Inhibition effect to HBsAg 9005784 6198 5792 0.498265 0.462353 0.497571 0.486063 0.893477 0.5139370.634835847 300 7150 8534 8318 0.379771 0.259715 0.278452 0.305980.4074138 1.207957 0.252210647 100 9830 11212 10210 0.147294 0.0274120.11433 0.096345 0.101434 2.111612 0.04583464 33.33333 13942 12368 134780 0 0 0 0.0050891 3.111612 0.001632835 11.11111 12418 11634 11352 0 00.015267 0.005089 0.005089 4.106523 0.001237728 Control Cell Blank 0Dilution 3 IC50 611.0919568 Third batch: (rSIFN-co Inhibition effect toHBeAg 900 9702 9614 8110 0.428016 0.433204 0.521872 0.461031 1.3169830.538969 0.709599543 300 8914 10032 8870 0.4744723 0.40856 0.4770660.453366 0.8559525 1.085603 0.440859127 100 16312 12688 13934 0.0383210.251975 0.178517 0.156271 0.402586 1.929332 0.172641621 33.33333 1508012814 13288 0.110954 0.244547 0.216602 0.190701 0.2463153 2.7386310.082519158 11.11111 21928 15366 15728 0 0.094093 0.072751 0.00556150.055615 3.683017 0.014875633 Control Cell 17544 Blank 0 Dilution 3 IC50382.0496935 Inhibition effect to HBsAg 900 5616 6228 5346 0.4968640.442035 0.521054 0.486651 0.763125 0.513349 0.597838293 300 8542 85907096 0.234725 0.230425 0.364272 0.276474 0.2764738 1.236875 0.182690031100 11420 11360 11394 0 0 0 0 0 2.236875 0 33.33333 12656 11582 13110 00 0 0 0 0 11.11111 13142 12336 13342 0 0 0 0 0 4.236875 0 Control Cell11528 Blank 0 Dilution 3 IC50 694.7027149 HBeAg: Average IC50: 450.2434SD: 132.315479 HBsAg: Average IC50: 649.1894 SD: 42.29580

TABLE 4.2 Results of inhibition rate of Intron A(IFN-α2b) to HBsAg andHBeAg Inhibition rate Average Accumulated Concentration First SecondThird First Second Third inhibition Accumula- 1- inhibition (×10⁴ IU/ml)well well well well well well rate tion Accumulation rate Inhibitioneffect to HBeAg 300 14918 11724 9950 0 0.029711 0.176529 0.0687470.068747 0.931253 0.068746724 100 14868 16890 15182 0 0 0 0 0 1.931253 0 33.33333 16760 21716 16400 0 0 0 0 0 2.931253 0  11.11111 20854 1504216168 0 0 0 0 0 3.931253 0  3.703704 12083 12083 12083 0 0 0 0 04.931253 0 Control Cell 17544 Blank 0 Dilution 3 IC50 FALSE Inhibitioneffect to HBsAg 300 9226 8196 9658 0.152489 0.247106 0.521054 0.17080.189295 0.8292 0.185857736 100 10946 10340 10828 0 0.050156 0.3642720.018495 0.0184947 1.810705 0.010110817  33.33333 12250 12980 13934 0 00 0 0 2.810705 0  11.11111 12634 12342 12000 0 0 0 0 0 3.810705 0 3.703704 10886 10886 10886 0 0 0 0 0 4.810705 0 Control Cell 10886Blank 0 Dilution 3 IC50 FALSE

TABLE 4.3 Results of inhibition rate of Infergen ® to HBsAg and HBeAgInhibition rate Concentration First Second Third First Second ThirdAverage Accumulated (×10⁴ IU/ml) well well well well well wellinhibition rate Accumulation 1-Accumulation inhibition rate First batch:(Infergen ®) Inhibition effect to HBeAg 900 14172 12156 17306 0.0916550.220869 0 0.104175 0.306157 0.895825 0.254710274 300 13390 12288 162520.1417767 0.212409 0 0.118062 0.2019827 1.777764 0.102024519 100 1436418834 14194 0.079349 0 0.090245 0.056531 0.083921 2.721232 0.02991667833.33333 15722 16034 16340 0 0 0 0 0.0273897 3.721232 0.00730659211.11111 17504 17652 14320 0 0 0.082169 0.02739 0.02739 4.6938430.005801377 Control Cell 15602 Blank 0 Dilution 3 IC50 FALSE Inhibitioneffect to HBsAg 900 12080 11692 12234 0 0.01275 0 0.00425 0.0251630.99575 0.024647111 300 12840 11484 12350 0 0.030313 0 0.0101040.0209125 1.985646 0.010422073 100 12894 14696 15086 0 0 0 0 0.0108082.985646 0.003606955 33.33333 15032 12928 13020 0 0 0 0 0.01080813.985646 0.002704416 11.11111 11794 11984 11508 0.004137 0 0.0282870.010808 0.010808 4.974837 0.002167838 Control Cell 11843 Blank 0Dilution 3 IC50 FALSE Second batch: (Infergen ®) Inhibition effect toHBeAg 900 6278 6376 6408 0.200051 0.187564 0.183486 0.190367 0.2746350.809633 0.253290505 300 7692 9092 6394 0.0198777 0 0.18527 0.0683830.0842678 1.74125 0.046161005 100 8960 7474 8190 0 0.047655 0 0.0158850.015885 2.725365 0.005794856 33.33333 8530 8144 9682 0 0 0 0 0 3.7253650 11.11111 7848 7848 7848 0 0 0 0 0 4.725365 0 Control Cell 7848 Blank 0Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 12364 12268 122740.036171 0.043655 0.043187 0.041004 0.140162 0.958996 0.12751773 30011590 12708 13716 0.0965076 0.009355 0 0.035287 0.0991581 1.9237090.0490186 100 12448 13468 13982 0.029623 0 0 0.009874 0.063871 2.9138340.02144964 33.33333 12616 11346 12444 0.016526 0.115529 0.0299350.053996 0.0539965 3.859838 0.013796309 11.11111 12828 12828 12828 0 0 00 0 4.859838 0 Control Cell 12828 Blank 0 Dilution 3 IC50 FALSE Thirdbatch: (Infergen ®) Inhibition effect to HBeAg 900 7240 6642 61580.064599 0.14186 0.204393 0.136951 0.217399 0.863049 0.201211735 30011072 8786 6902 0 0 0.108269 0.03609 0.0804479 1.82696 0.042176564 1007016 9726 7552 0.09354 0 0.024289 0.039276 0.044358 2.787683 0.01566301733.33333 7622 8866 8676 0.015245 0 0 0.005082 0.0050818 3.7826010.001341671 11.11111 7740 7740 7740 0 0 0 0 0 4.782601 0 Control Cell7740 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 1104811856 11902 0.04775 0 0 0.015917 0.015917 0.984083 0.015916796 300 1345412896 11798 0 0 0 0 0 1.984083 0 100 12846 13160 12546 0 0 0 0 02.984083 0 33.33333 12680 12458 12360 0 0 0 0 0 3.984083 0 11.1111111602 11602 11602 0 0 0 0 0 4.984083 0 Control Cell 11602 Blank 0Dilution 3 IC50 FALSE HBeAg: Average IC50: 0 SD: 0 HBsAg: Average IC50:0 SD: 0

Example 7 The Clinic Effects of Recombinant Super-Compound Interferon(rSIFN-co)

The recombinant super-compound interferon (rSIFN-co) is an invention forviral disease therapy, especially for hepatitis. Meanwhile, it caninhibit the activity of EB viruses, VSV, Herpes simplex viruses,cornaviruses, measles viruses, et al. Using Wish cells/VSV system as theassay for anti-virus activity, the results showed that: the other rIFN,was 0.9×10⁸ IU/mg, Intron A was 2.0×10⁸ IU/mg and rSIFN-co was 9×10⁸IU/mg. The anti-viral activity of rSIFN-co is much higher than those ofthe former two.

Under the permission of the State Food and Drug Administration (SFDA),People's Republic of China, the clinical trials have taken place in WestChina Hospital, Sichuan University, the Second Hospital of ChongqingMedical University, the First Hospital of School of Medical, ZhejiangUniversity since the February 2003. The clinical treatment which focuseson hepatitis B is conducted under the guidance of the mutilcenter,double-blind random test. IFN-α1b was used as control, and the primaryresults showed the following:

The Effect of rSIFN-co Compared with IFN-α1b in the Treatment of ChronicActive Hepatitis B

1. Standard of Patients Selection:

Standards 1-4 are effective for both treatment with rSIFN-co (9 μg) andIFN-α1b (5 MU, 50 μg), and Standard 1-5 are for rSIFN-co (15 μg)treatment.

1). Age: 18-65

2). HBsAg-test positive over last six months, HBeAg-test positive, PCRassay, HBV-DNA copies ≧10⁵/ml3). ALT ≧two times the normal value4). Never received IFN treatment; or received the Lamividine treatmentbut failed or relapsed5) Once received other IFNs (3 MU or 5 MU) treatment six months agofollowing the standard of SFDA, but failed or relapsed

2. Evaluation of the Effects:

In reference to the recommendations from the Tenth China NationalCommittee of Virus Hepatitis and Hepatopathy, the effects were dividedinto three degrees according to the ALT level, HBV-DNA and HBeAg tests.

-   Response: ALT normal level, HBV-DNA negative, HBeAg negative-   Partial response: ALT normal level, HBV-DNA or HBeAg negative-   Non response: ALT, HBV-DNA and HBeAg unchanged-   The response and partial response groups were considered effective    cases.

3. Results of Clinic Trial:

Group A: treatment with rSIFN-co (9 μg)

Group B: treatment with IFN-α1b (5 MU, 50 μg)

HBsAg HBeAg HBV-DNA Transfer Transfer Transfer Heptal to to to functionEffective negative negative negative Recovery Period group Medicinecases Rate rate rate rate rate 8-12 A rSIFN- 32 46.88 9.38 28.13 37.5084.38 week co(9 μg) (15)   (3)   (9)   (12)   (27)   B IFN-α1b 32 21.880.00  9.38 15.63 56.25 (5MU, 50 μg) (7)   (0)   (3)   (5)   (18)   16-24A rSIFN- 64 54.69 7.81 25.00 34.38 90.63 week co(9 μg) (35)   (5)  (16)   (22)   (58)   B IFN-α1b 64 25.00 0.00  9.38 18.75 78.13 (5MU 50μg) (16)   (0)   (6)   (12)   (50)  

In Group C, the cases were prior treatment of chronic active hepatitis Bwith other IFNs (3 MU or 5 MU) that failed or relapsed and then weretreated with rSIFN-co (15 μg), subcutaneous injection, every one day,for 24 weeks. The total cases were 13. After 12 weeks treatment, 7 of 13(53.85%) were effective. 3 of 13 (23.08%) HBeAg transferred to negative;7 of 13 (53.85%) HBV-DNA transferred to negative; 11 of 13 (84.62%)heptal functions recovered to normal.

4. The Side Effects of rSIFN-co Compared with IFN-α1b in the Treatment

The side effects of IFN include fever, nausea, myalgia, anorexia, hairloss, leucopenia and thrombocytopenia, etc. The maximum dose of IFN-α1bis 5 MIU per time; the routine dose is 3 MIU. When taken the routinedose, 90% patients have I-II degree (WHO standard) side effects. Theyhad fever lower than 38° C., nausea, myalgia, anorexia, etc. When takenat maximum dose, the rate of side effects did not rise obviously, butwere more serious. The maximum dose of rSIFN-co is 24 μg, subcutaneousinjection, every one day for 3 months. The routine dose is 9 μg. Whenroutine doses were used, less than 50% of patients had I-II degree (WHOstandard) side effects, including fever below 38° C., nausea, myalgia,anorexia, leucopenia and slight thrombocytopenia. With maximum dosage,about 50% patients suffered from leucopenia and thrombocytopenia afterusing rSIFN-co one month, but those side effects disappeared afterstopping treatment for one week. It is safe for continued use.

The Observations of rSIFN-co Treat Hepatitis C

1. Standard of Patients Selection

1) age: 18-652) HCV antibody positive3) ALT≧1.5 times of the normal value, last more than 6 months

2. Evaluation of the Effects:

Referring to the standard of Infergen® for treatment of hepatitis C andaccording to the ALT level and HCV-RNA test, divided the effects intothree degree:

Response: ALT normal level, HCV-RNA negative

Partial response: ALT normal level, HCV-RNA unchanged

Non response: ALT and HCV-RNA unchanged

3. Effects in Clinic

The clinical trial was done at the same time with hepatitis B treatment.46 cases received the treatment, 9 μg each time, subcutaneous injection,every day for 24 weeks. After treatment, 26 of 46 (56.52%) have obviouseffects, 12 of 46 (26.09%) HCV-RNA transferred to negative, 26 of 46(56.52%) heptal functions recovered to normal.

Example 8 Comparison of Inhibitory Effects of Different Interferons onHBV Gene Expression

Hepatitis B virus (HBV) DNA contains consensus elements fortransactivating proteins whose binding activity is regulated byinterferons. Treatment of HBV-infected hepatocytes with interferonsleads to inhibition of HBV gene expression. The aim of the present studywas to characterize the effects of different interferons on HBVregulated transcription. Using transient transfection of human hepatomacells with reporter plasmids containing the firefly luciferase geneunder the control of HBV-Enhancer EnH I, Enh II and core promoter,Applicant studied the biological activities of three differentinterferons on transcription.

Materials and Methods

1. Interferons: IFN-con1 (Infergen®), IFN-Hui-Yang (rSIFN-co) andIFNα-2b (Intron A).2. Reporter plasmid: The DNA fragments containing HBV-Enhancer EnH I,Enh II and core promoter were prepared using PCR and blunt-end clonedinto the Smal I site of the promoter- and enhancer-less fireflyluciferase reporter plasmid pGL3-Basic (Promega, WI, USA). The resultingreporter plasmid was named as pGL3-HBV-Luc.3. Cell Culture and DNA transfection: HepG2 cells were cultured in DMEMmedium supplemented with 10% FBS and 100 U/ml penicillin and 100 ug/mlstreptomycin. The cells were kept in 30° C., 5% CO2 incubator. The cellswere transfected with pGL3-HBV-Luc reporter plasmid using Boehringer'sLipofectin transfection kit. After 18 hours, the medium containingtransfection reagents was removed and fresh medium was added with orwithout interferons. The cells were kept in culture for another 48hours.4. Luciferase Assay: Forty-eight hours after addition of interferon, thecells were harvested and cell lysis were prepared. The proteinconcentration of cell lysates were measured using Bio-Rad Protein Assaykit. The luciferase activity was measured using Promega's LuciferaseReporter Assay Systems according to the instructions of manufacturer.

Results Expression of Luciferase Activity in DifferentInterferon-Treated Cell Lysates

No treatment IFN-con1 rSIFN-co IFNα-2b 100 65 32 73

This result shows that rSIFN-co inhibits most effectively on theexpression of HBV gene expression of HB core Antigen. This data showsinhibitory effect of rSIFN-co is twice better than Infergen® and IntronA. See FIG. 10.

Example 9 Recombinant Super-Compound Interferon Spray

Major component: Recombinant Super Compound Interferon

Characteristic: Liquid, no insoluble material

Pharmacology: Recombinant Super-Compound Interferon has a wide spectrumof anti-virus activity. Its effects are 5-20 times higher than thoseinterferons (IFNs) which are available on the market. It can inhibitcoronavirus growth in cell culture. In vitro test shows that rSIFN-cohas an obvious anti-SARS virus activity. rSIFN-co effect to 10,000 and1000 TCID₅₀. The Inhibitory Indexes are 0.92 μg/ml and 0.18 μg/mlrespectively. The Treatment Indexes (TI) are 151.28, 773.22respectively. The mechanism is interruption of the combination reactionbetween the IFN and the correspondent receptor, and inducement of theexpression of 2′5′-A synthesizenzyme, protein kinase R in the targetcell, therefore inhibiting expression of the viral protein. IFN caninduce expression of various anti-virus proteins to inhibit thereproduce of viral proteins, enhance the function of Natural Killer (NK)cell and other Immune regulative functions, and inhibit the invasion ofviruses.

Acute toxicity: All mice are alive after the maximum dose (1000 times tohuman dose) subcutaneous injection, did not observe LD50.

Indication: Prevention of Severe Acute Respiratory Syndrome

Dosage and Administration: Spray to both nasal cavity and throat, threetimes a day.

Adverse reactions: There was no report of adverse reactions from therSIFN-co spray. It did not induce allergy. If the stimulation isoccasional, adverse gastrointestinal reaction is small, and no otherobvious adverse reaction was noted during treatment, it is safe tocontinue use. All reactions will resolve themselves.

Warning: Patients allergic to IFN and productions of E. Coli. cannot usethis product.

Precautions: Before first use, spray twice to expel the air. If there isany cloudy precipitation material, if the product is expired, or thereis material on the vial, do not use it.

Pediatric Use: It is unclear.

Geriatric Use: It is unclear.

Nursing mothers and pregnant women: Use with care or under physician'ssupervision.

Drug Interactions: It is unclear.

Overdose: One-time dose of over 27 million of International Units havenot produced any adverse effects.

Supplied: 1 spray/pack, 20 ug (1×10⁷ IU)/3 ml. See FIGS. 11A-11D.

Storage: Store at 4-8° C. Do not freeze, protect from light.

Effective period: Approximately one year

Manufacture: Manufactured by Sichuan Huiyang life-engineering Ltd.

Address: 8 Yusa Road, Room 902, Building A

-   -   Chengdu, 610017    -   Sichuan, P.R. China

Example 9-A In Vitro Effect of a New-Style Recombinant CompoundInterferon on SARS-Associated Coronavirus

Sample supplied by: Huiyang Life Engineering Lt Company, SiChuanProvince

Experimenter: Molecular Biology Department, microorganism andepidemiology Institute, Academy of Military Medical Science

Original data: Preserved in archive of Molecular Biology Department,microorganism and epidemiology Institute, Academy of Military MedicalScience

1. Materials

Medicine: New-type recombinant compound interferon, 9 μg each, suppliedby Huiyang Life Engineering Lt Company, SiChuan Province, Lot number:20020501.

Cells: Vero E₆, supplied by Molecular Biology Department ofMicroorganism and Epidemiology Institute, Academy of Military MedicalScience.

Virus: SARS-associated coronavirus, BJ-01, supplied by Molecular BiologyDepartment of Microorganism and Epidemiology Institute, Academy ofMilitary Medical Science.

Cell medium: DMEM supplemented with 10% FBS.

2. Condition

Virus was measured in grade 3^(rd) laboratory of biosafety

3. Method

CPE (cytopathic effect) assay of TCID₅₀: 100 μl of Vero E₆ cells wereplated in 96-well plates at 2×10⁴ cells per well. After 24 hr incubationat 37° C., Vero E6 monolayer cells were treated with 9 levels ofSARS-associated coronavirus dilution by 10-fold dilution, 4 wells perdilution. The cells were incubated at 37° C. and 5% CO₂. CPE (cytopathiceffect) was examined daily by microscopy. CPE less than 25% wasdetermined as +, 26-50% as ++, 51-75% as +++, 76-100% as ++++. CPE wasrecorded. Then TCID₅₀ was calculated by Reed-Muench method.

Cytotoxicity of medicine: Vero E₆ cells were inoculated into 96-wellplates at 2×10⁴ cells (100 ul) per well. After 24-hr incubation at 37°C., cells grew up to monolayer. The medicine was diluted into 36, 18, 9,4.5, 2.259 μg/ml (final concentration) and added into wells each for 4wells. The normal cells as control group were set. CPE of medicine groupwas daily observed during 5-day period, and then the concentration ofmedicine exhibiting no toxicity was determined.

CPE assay of the activity of the medicine against SARS-associatedcoronavirus: 100 μl of Vero E₆ cells were plated in 96-well plates at2×10⁴ cells per well. After 24 hr incubation at 37° C., cells grew up tomonolayer. The medicine at the maximal concentration exhibiting nocytotoxicity was diluted into 5 levels by 2-fold dilution and added intowells (100 μl per well). By incubation with 5% CO₂ at 37° C. for24-hour, different concentration of virus (10⁻³, 10⁻⁴, 10⁻⁵) were added.After treatment with virus for 48-72 hours, CPE was examined (CPE lessthan 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100% as++++, normal cell as −). The cells were divided into the normal group,the medicine control group, and the different dilution of virus controlgroup, 4 wells per group. CPE was examined daily. Till cytopathic effectwas obviously exhibited in the virus control group, the anti-virusactivity of interferon was evaluated. The experiment was repeated. IC₅₀of the medicine was calculated by Reed-Muench method.

4. Results

Toxicity of virus: TCID₅₀ of virus was 10⁻⁸.

Cytotoxicity of medicine: the concentration of Recombinant compoundinterferon exhibiting no cytotoxicity was 18 μg/ml, the cells shape wassimilar with the control group, and no cytopathic effect was exhibited.

The anti-virus effect of the medicine: Shown in Table 9-A.1 and Table9-A.2

TABLE 9 A.1, the anti-virus effect of new-type recombinant compoundinterferon (first experiment) Concentration of CPE at differentconcentration IFN of virus (μg/ml) 10⁻³ 10⁻⁴ 10⁻⁵ 18 − − − 9 − − −4.5 + + − − 2.25 + + + + + − 1.125 + + + + + + + + + + Viruscontrol + + + + + + + + + + + group Normal group − − − Medicine control− − − group

TABLE 9 A.2, the anti-virus effect of new-type recombinant compoundinterferon (second experiment) Concentration of CPE at differentconcentration IFN of virus (μg/ml) 10⁻³ 10⁻⁴ 10⁻⁵ 18 − − − 9 − − − 4.5 +− − 2.25 + + + + + − 1.125 + + + + + + + + + + Viruscontrol + + + + + + + + + + + + group Normal group − − − Medicinecontrol − − − group

5. Conclusion

The concentration of the new-type recombinant compound interferonexhibiting no cytotoxicity at 18 μg/ml. Its IC₅₀ were 1.27, 2.25, and4.04 μg/ml respectively according to the concentration of 10⁻⁵(1000TCID₅₀), 10⁻⁴ (1000TCID₅₀), 10⁻³ (100000TCID₅₀) of SARS-associatedcoronavirus (Table 9-A.3).

TABLE 9 A.3, IC₅₀ of IFN at different concentrations of virus Dilutionof virus IC50 of IFN (ug/ml) 10⁻³ 4.04 10⁻⁴ 2.25 10⁻⁵ 1.27

Principal: Jin-yan Wang

Laboratory assistant: Yan-hong Zhao, Xiao-guang Ji, Xiao-yu Li.

Original data: Preserved in archives of Molecular Biology Department,microorganism and epidemiology Institute, Academy of Military MedicalScience

Date: From May 12th to 30th, 2003

Example 9-B In Vitro Effect of a New-Type Recombinant CompoundInterferon and Recombinant Interferon α-2b Injection on SARS-AssociatedCoronavirus

Sample (rSIFN-co) supplied by: Huiyang Life Engineering Ltd., Sichuanprovince

Experimenter: Molecular Biology Department, Institute of microbiologyand epidemiology, Academy of Military Medical Science

Original data: Preserved in monument room of Molecular BiologyDepartment, Institute of microbiology and epidemiology, Academy ofMilitary Medical Science

1. Materials

Medicine: New-type recombinant compound interferon (rSIFN-co), 618μg/ml, supplied by Huiyang Life Engineering Ltd., SiChuan Province;Alfaron (recombinant interferon α-2b injection), supplied by TianjinHualida Biotechnology Co., Ltd. 30 ug/vial (300,0000 IU/vial), LotNumber: 20030105.

Cells: Vero E₆, supplied by Molecular Biology Department of Institute ofmicrobiology and epidemiology, Academy of Military Medical Science.

Virus: SARS-associated coronavirus, BJ-01, supplied by Molecular BiologyDepartment of Institute of microbiology and epidemiology, Academy ofMilitary Medical Science.

Condition: Viruses were measured in grade 3^(rd) laboratory of biosafety

2. Method

TCID₅₀ was measured with CPE assay: Vero E₆ cells were inoculated in96-well plates at 2×10⁴ cells (100 μl) per well. After a 24-hrincubation at 37° C., Vero E6 monolayers were treated with 9 levels ofSARS-associated coronavirus dilution by 10 times decreasing, eachdilution per 4 wells. The cells were incubated at 37° C. and 5% carbondioxide. CPE was examined daily by phase-contrast microscopy. CPE lessthan 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100% as++++. CPE was recorded. Then TCID₅₀ was calculated by Reed-Muenchmethod.

TC₅₀ of IFNs were measured by MTT assay: Vero E₆ cells were inoculatedin 96-well plates at 2×10⁴ cells per well (100 μl). After 24-hrincubation at 37° C., the supernatant liquid was removed when cells grewup to monolayer, then Vero E₆ was treated with different concentrationof IFNs, each dilution per 4 wells. Normal group was set. After 5-dayobservation, the cells were mixed with MTT for 4 hours. After that,remove the liquid, and then thereafter DMSO were added into cells for0.5 hour. The OD_(570nm) was measured by microplate reader. Finally,TC₅₀ was calculated by Reed-Muench method.

The activity of the INFs against SARS-associated coronavirus wasmeasured with MTT assay: 100 μl of Vero E₆ cells were inoculated in96-well plates at 2×10⁴ cells per well. After 24-hr incubation 37° C.,cells became monolayer. The medicine dilution at the concentration ofexhibiting no cytotoxicity was 5 times decreasing and there were 5levels of dilution. Then each dilution was added to 4 wells, 100 ul perwell. After 24-hour incubation at 37° C. and 5% CO₂, IFN solution wasremoved, then different concentrations of virus dilution (10000, 1000,100 TCID₅₀) were added into dishes, 4 wells per dilution. The cells weredivided into the normal group, the medicine control group, and thedifferent dilution of virus control group (10000, 1000, 100 TCID₅₀). Thecells were incubated at 37° C. and 5% CO₂ for 48-72 hr, until cytopathiceffect was exhibited in the virus control group, CPE was recorded (CPEless than 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100%as ++++, normal cell as −). The growth ability of cells was measuredwith MTT assay, and then the antivirus effect of the INFs was evaluated.The experiment was repeated 3 times. IC₅₀ of the medicine was calculatedby Reed-Muench method.

3. Results

TCID₅₀ of virus: TCID₅₀ of virus was 10⁻⁷.

TC₅₀ of IFNs: The concentration of new-type recombinant compoundinterferon (rSIFN-co) exhibiting no cytotoxicity was 100 μg/ml, and thatof recombinant IFNα-2b was 12.5 μg/ml, the cells shape was identicalwith the normal group at that concentration. TC50 of new-typerecombinant compound interferon (rSIFN-co) was 139.18 μg/ml, that ofrecombinant IFNα-2b was 17.18 μg/ml.

TABLE 9 B.1 TC₅₀ of IFNs TC₅₀ (μg/ml) 1^(st) 2^(nd) 3^(rd) Mean value-IFN experiment experiment experiment (X ± SD, n = 3) new-type 141.42125.96 150.08 139.18 ± 12.22 recombinant compound interferon IFNα-2b17.68 15.75 18.10  17.18 ± 1.25

The anti-virus effect of the medicine: The anti-virus effects of twoIFNs were observed in vitro. The results of the experiments are shown onthe Table 9-B.2, and the results of TI are shown on the Table 9-B.3.

TABLE 9 B.2, The anti-virus activity of IFNs Concentration IC₅₀ (μg/ml)of 1^(st) 2^(nd) 3^(rd) Mean value- IFNs virus (TCID₅₀) experimentexperiment experiment (X ± SD, n = 3 new-type 10000 0.79 1.04 0.93 0.92± 0.12 recombinant compound interferon IFNα-2b 5.04 4.56 4.65 4.75 ±0.25 new-type 1000 0.19 0.18 0.18 0.18 ± 0.01 recombinant compoundinterferon IFNα-2b 1.18 1.19 1.12 1.16 ± 0.04 new-type 100 0.08 0.100.11 0.10 ± 0.02 recombinant compound interferon IFNα-2b 0.33 0.21 0.300.28 ± 0.06

TABLE 9 B.3, The anti-virus activity of IFNs Concentration of TI IFNsvirus (TCID₅₀) TC₅₀ (μg/ml) IC₅₀ (μg/ml) (TC₅₀/IC₅₀) new-type 10000139.18 0.92 151.28 recombinant compound interferon IFNα-2b 17.18 4.753.62 new-type 1000 139.18 0.18 773.22 recombinant compound interferonIFNα-2b 17.18 1.16 14.78 new-type 100 139.18 0.10 1391.80 recombinantcompound interferon IFNα-2b 17.18 0.28 61.36

4. Conclusion

The protection effect of new-type recombinant compound interferon(rSIFN-co) and IFNα-2b on Vero E₆ was observed in vitro, and theanti-virus ability of IFNs was manifested. IC₅₀ of new-type recombinantcompound interferon on SARS-associated coronavirus at the concentrationof 10000, 1000, and 100 was 0.92, 0.18, and 0.10 μg/ml in threeexperiments, TI of that was 151.28, 773.22, and 1391.80 respectively.IC₅₀ of IFNα-2b was 4.75, 1.16, and 0.28 μg/ml, TI (treatment index) ofthat was 3.62, 14.78, 61.36 respectively.

Most importantly, the two tests (See the above Examples 9A & 9B) of invitro anti-SARS virus effect of rSIFN-co all testified that even theeffective dose of rSIFN-co to inhibit SARS virus is 1/5 of that ofInterferon α-2b which was used clinically in China at present, theTreatment Index (TI) of rSIFN-co is nearly 50 times of that ofInterferon a-2b. (SEE: In vitro effect of a new-type recombinantcompound interferon and recombinant interferon-α-2b injection onSARS-associated coronavirus. By The Institute of Microbiology &Epidemiology, Academy of Military Medical Science) Also, see FIG. 12.

Thirty thousand sprays of rSIFN-co had been used among front-line nursesand doctors, and people at high risk in Sichuan province. The resultshows that none of the nurses and doctors infected SARS in SichuanProvince.

Principal: Jin-yan Wang

Laboratory assistant: Yan-hong Zhao, Xiao-guang Ji, Min Zhang, Jing-hua,Zhao.

Date: From July 1st to 30th, 2003

Example 10 Side Effects and Changes in Body Temperature when UsingrSIFN-co

There are usually more side effects to using interferon. The sideeffects includes: nausea, muscle soreness, loss of appetite, hair loss,hypoleucocytosis (hypoleukmia; hypoleukocytosis; hypoleukia), anddecrease in blood platelet, etc.

Method

Sample patients are divided into two groups. 10 patients in Group A wereinjected with 9 μg rSIFN-co. 11 patients in Group B were injected with 9μg Infergen®. Both groups were monitored for 48 hours after injections.First monitoring was recorded 1 hour after injection. After that,records were taken every 2 hours.

Table 11.1 is the comparison of side effects between patients beinginjected with 9 μg of rSIFN-co and 9 μg of Infergen®.

TABLE 11.1 Side Effects rSIFN-co Infergen ® 9 μg (Group A) 9 μg (GroupB) Person: n = 10 Person: n = 11 Body Systems Reactions HeadcountHeadcount In General Feeble 3 3 Fever 3 6 Sole heat 1 frigolabile 3 4Leg 3 strengthless Mild lumbago 2 1 Body soreness 4 5 Central NervousHeadache 3 6 System/Peripheral Dizziness 2 11 Nervous System Drowsiness3 Gastroenterostomy Apoclesis 1 Celiodynia 1 Diarrhea 1 MusculoskeletalMyalgia 1 2 system Arthralgia 2 Respiratory Stuffy nose 1 systemParopsia Swollen Eyes 1

Results

For those patients who were injected with rSIFN-co, the side effectswere minor. They had some common symptoms similar to flu, such as:headache, feebleness, frigolability, muscle soreness, hidrosis,arthralgia (arthrodynia; arthronalgia). The side effects of thosepatients whom were injected with Infergen® were worse than thoseinjected with rSIFN-co.

From FIGS. 13A-1, 13A-2, 13B-1, and 13B-2, it was obvious that the bodytemperatures of sample patients in Group B were higher than the patientsin Group A. It also reflected that the endurance of rSIFN-co was muchbetter than Infergen®.

Example 11 Effects of Recombinant Super-Compound Interferon (rSIFN-co)on Ebola Virus

Background: Ebola virus is a notoriously deadly virus that causesfearsome symptoms, the most prominent being high fever and massiveinternal bleeding. Ebola virus kills as many as 90% of the people itinfects. It is one of the viruses capable of causing hemorrhagic(bloody) fever. There is no specific treatment for the disease.Currently, patients receive supportive therapy. This consists ofbalancing the patient's fluids and electrolytes, maintaining theiroxygen level and blood pressure, and treating them for any complicatinginfections. Death can occur within 10 days of the onset of symptoms.

1. Materials

-   -   1.1 Drugs: rSIFN-co, provided by Sichuan Biotechnology Research        Center.    -   1.2 Virus: Ebola, supplied by The Academy of Military Medical        Science, Institute of Microbiology Epidemiology.    -   1.3 Safety level of experiment: Viral experiments were carried        under Biological Laboratory Safety System level 3.    -   1.4 Animals: 60 BALB/c mice

2 Method

-   -   2.1 60 mice were randomly separated into 6 groups, each group        consisting of 10 mice. Group 1 was treated with 1 μg/ of        rSIFN-co on the day of inoculation with Ebola virus. Group 2 was        treated with 1 μg/ of rSIFN-co day one (1) after inoculation        with Ebola virus. Group 3 was treated with 1 μg/ of rSIFN-co on        the day two (2) after inoculation with Ebola virus. Group 4 was        treated with 1 μg/ of rSIFN-co on day three (3) after        inoculation with Ebola virus. Group 5 was treated with 1 μg/ of        rSIFN-co on day four (4) after inoculation with Ebola virus.        Group 6 was not treated with rSIFN-co and this is designated as        the control group.    -   2.2 Administration of the medication: 1 μg/ of rSIFN-co was        administered once a day for six (6) consecutive days.

3 Results

-   -   All ten (10) mice in group 6 (control group) died. All mice in        groups one (1), two (2) and three (3) survived with no        observable toxic effect. In groups four (4) and five (5), showed        some effects.

4 Conclusion

-   -   Clearly these result show effectiveness of rSIFN-co against        Ebola virus.

Example 12 Anti-HIV Effects of Recombinant Super-Compound Interferon(rSIFN-co) 1. Materials

-   -   1.1 Wild-Type HIV    -   1.2 Drug Resistant HIV    -   1.3 293-CD4-CCR5 cells    -   1.4 DMEM, Gibco    -   1.5 Fetal Bovine Seru, Gibco    -   1.6 rSIFN-co provided by Sichuan Biotechnology Research Center

1.7 96-well plate, NUNC

-   -   1.8 CO₂ incubator    -   1.9 Laminar Flow Hood    -   1.10 Fluorometer    -   1.1 IU V Absorbance Meter    -   1.12 Others

2. Method

-   -   2.1 293-CD4-CCR5 cells in exponential (log) phase were obtained,        digested with 0.25% pancreatin, stained with Trypan blue stain        to determine cell number and diluted with DMEM to concentration        of 2.0×10⁵ cells per milliliter (cell/ml).    -   2.2 Each well of 96-well plate was filled with 100 μl        (microliters) of 293-CD4-CCR5-DMEM suspension solution. The        plate was placed into 5% carbon dioxide incubator at 37 degrees        Celsius and observed the next day seventy percent (70%) of basal        area of the well were recovered.    -   2.3 After supernatant was removed, 100 μl (microliters) of        different concentrations of rSIFN-co were added to each well.        Two controls were used: Phosphate Buffered Saline (PBS) and        Growth Media.    -   2.4 The plate was placed into carbon dioxide incubator at 37        degrees Celsius for approximately 18 to 20 hours.    -   2.5 Experimental wells: Different concentrations of the        Wild-Type HIV and Drug Resistant HIV viruses were placed into        each well at 100 μl (microliters) per well.    -   Control wells: No virus was added, only 100 μl (microliters) of        DMEM per well.    -   2.6 The plate was placed into carbon dioxide incubator at 37        degrees Celsius for approximately 24 hours.    -   2.7 Routine Luciferase assay was performed and protein        concentrations of the supernatants were measured. Luciferase was        measured in RLU/mg units.

3 Results

-   -   rSIFN-co can inhibit HIV at level of ≧4 nanograms per milliliter        (ng/ml). See Table 4 and FIGS. 14-15. When using Luciferase as Y        axis and concentration of rSIFN-co as X axis, using EXCEL, it is        clear that at level of rSIFN-co ≧4 nanograms per milliliter        (ng/ml), the level of Luciferase activities are obviously lower        than in PBS and Medium controls. A clear inverse dose-dependent        response has been shown.

TABLE 4 Comparison of Inhibition of Wild-Type HIV and Drug Resistant HIVby rSIFN-co Concentration Luciferase Assay of rSIFN-co Wild-Type HIVDrug Resistant HIV Medium 13500 + 2000 18000 + 2000   1 μg/ml  3000 +200  2800 + 800  500 ng/ml  3000 + 600  2800 + 900  250 ng/ml  3400 +400  4000 + 600  125 ng/ml  4300 + 200  4100 + 600 62.5 ng/ml  4300 +400  4100 + 1000   31 ng/ml  5000 + 800  5100 + 800   15 ng/ml  7200 +400  6000 + 1500  7.5 ng/ml  7000 + 800  7700 + 1300   4 ng/ml  9000 +2000  8900 + 2000 PBS 13000 + 3000 15100 + 2300 Medium 16000 + 360019000 + 2500 4 Conclusion: rSIFN-co is effective against both: Wild-TypeHIV and Drug Resistant HIV.

Example 13 Anti-Influenza Effects of Recombinant Super-CompoundInterferon (rSIFN-co) 1. Materials

-   -   1.1. 10-day old chick embryonic membrane cells    -   1.2. SIFN-co provided by Sichuan Biotechnology Research Center    -   1.3. Influenza virus provided by Molecular Biology Department of        Institute of microbiology and epidemiology, Academy of Military        Medical Science.    -   1.4. DMEM, Gibco    -   1.5. Newborn Calf Serum    -   1.6. 96-well plate, NUNC    -   1.7. CO₂ incubator    -   1.8. Laminar Flow Hood    -   1.9. Inverted Microscope    -   1.10. Others

2. Method

-   -   2.1 10-day old chick embryonic membrane cell in exponential        (log) phase were obtained, digested with 0.25% pancreatin,        stained with Trypan blue stain to determine cell number and        diluted with DMEM to concentration of 2.0×10⁵ cells per        milliliter (cell/ml).    -   2.2 Each well of 96-well plate was filled with 100 μl        (microliters) of 293-CD4-CCR5-DMEM suspension solution. The        plate was placed into carbon dioxide incubator at 37 degrees        Celsius. The next day cells grew to a monolayer.    -   2.3 After supernatant was removed, 100 μl (microliters) of        different concentrations of rSIFN-co were added to each well.        Two control wells: No rSIFN-co was added    -   2.4 The plate was placed into carbon dioxide incubator at 37        degrees Celsius for approximately 18 to 20 hours.    -   2.5 Experimental wells: Different concentrations of the        Influenza virus were placed into each well at 100 microliters        (μl) per well.    -   Control wells: No Influenza virus was added, only 100 μl        (microliters) of DMEM per well.    -   2.6 The plate was placed into carbon dioxide incubator at 37        degrees Celsius for approximately 24 hours.    -   2.7 Cells were observed under inverted microscope.

3. Results

-   -   3.1 Under inverted microscope, the cells in the control well        with Influenza virus added and without interferon had obvious        CPE, such as rounding of cells, cell necroses, decrease in        reflective light and sloughing off.    -   3.2 Cells from the experimental wells containing rSIFN-co at        concentration ≧10 nanogram per milliliter (ng/ml) had no CPE and        morphology comparable to normal cells. See FIG. 16.    -   3.3 Control Wells without Influenza virus added and without        interferon did not have any CPE.

4 Conclusion

-   -   At concentration ≧10 nanogram per milliliter (ng/ml) rSIFN-co is        effective against Influenza virus.

1-70. (canceled)
 71. A method for treating tumors in a subject,comprising administering to the subject an effective amount of arecombinant interferon, wherein the interferon has the amino acidsequence of SEQ ID NO:2 and is encoded by the nucleotide sequence SEQ IDNO:1, wherein the tumors are selected from the group consisting of skincancer, basal cell carcinoma, liver cancer, thyroid cancer,rhinopharyngeal cancer, solid carcinoma, prostate cancer, esophagealcancer, pancreatic cancer, superficial bladder cancer, hemangioma,epidermoid carcinoma, cervical cancer, glioma, leucocythemia, acuteleucocythemia, chronic leucocythemia, lymphadenoma, and polycythemiavera.
 72. The method of claim 71, wherein the effective amount of therecombinant interferon comprises a dose in a range from nanogram tomicrogram.
 73. The method of claim 71, wherein the interferon isadministered orally, via vein injection, muscle injection, peritonealinjection, subcutaneous injection, nasal or mucosal administration, orby inhalation.
 74. The method of claim 71, wherein the interferon isadministered subcutaneously or intramuscularly at a dose of higher thanor equal to 10 Million International Units per square meter of surfacearea.
 75. The method of claim 71, wherein the interferon is administeredsubcutaneously or intramuscularly at a dose of higher than or equal to20 Million International Units per square meter of surface area.
 76. Themethod of claim 71, wherein the interferon is lyophilized.
 77. Themethod of claim 71, wherein the interferon is administered following aprotocol of 9 μg, 15 μg or 24 μg per day, 3 times a week, for a total of24 weeks.